Microbial production of rotundone

ABSTRACT

The present disclosure provides methods and compositions for producing rotundone. In various aspects, the present disclosure provides enzymes, polynucleotides encoding said enzymes, and recombinant microbial host cells (or microbial host strains) for the production of rotundone. In some embodiments, the present disclosure provides microbial host cells for producing rotundone at high purity and/or yield, from either enzymatic transformation of α-guaiene, or from sugar or other carbon source. The present disclosure further provides methods of making products containing rotundone, including flavor or fragrance products, among others.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/727,815, filed Sep. 6, 2018, which is hereby incorporated byreference in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename: MAN-019PC SequenceListing_ST25; date recorded: Sep. 6, 2019; file size: 235,920 bytes).

BACKGROUND

Rotundone is an oxygenated sesquiterpene (sesquiterpenoid) that isresponsible for a pleasing spicy, ‘peppery’ aroma in various plants,including grapes (especially syrah or shiraz, mourvèdre, durif,vespolina, and grüner veltliner varietals), and a large number of herbsand spices, such as, e.g., black and white pepper, oregano, basil,thyme, marjoram, and rosemary. Given its aroma, rotundone is anattractive molecule for applications in fragrances and flavors.

α-Guaiene is the precursor to (−)-rotundone. α-Guaiene is asesquiterpene hydrocarbon found in oil extracts from various plants, andis converted to (−)-rotundone by aerial oxidation or enzymatictransformation.

Given the commercial value of rotundone, cost effective, scalable,and/or sustainable processes for its production are needed.

SUMMARY

In various aspects, the present disclosure provides methods andcompositions for producing rotundone. In various aspects, the presentdisclosure provides enzymes, polynucleotides encoding said enzymes, andrecombinant microbial host cells (or microbial host strains) for theproduction of rotundone. In some embodiments, the present disclosureprovides microbial host cells for producing rotundone at high purityand/or yield, from either enzymatic transformation of α-guaiene, or fromsugar or other carbon source. The present disclosure further providesmethods of making products containing rotundone, including flavor orfragrance products, among others.

In some embodiments, the present disclosure provides a microbial hostcell expressing an enzyme pathway catalyzing the conversion of farnesyldiphosphate (FPP) to rotundone, the enzymatic pathway comprising anα-guaiene terpene synthase enzyme (αGTPS) and an α-guaiene oxidase(αGOX) enzyme. In these embodiments, the microbial cells can synthesizerotundone product from any suitable carbon source. In some embodiments,the specificity of the α-guaiene synthase enzyme enables production ofrotundone at high yield with fewer terpenoid byproducts. In someembodiments, the αGOX produces rotundone as the predominant oxygenatedproduct.

In some embodiments, the microbial host cell further expresses oroverexpresses one or more enzymes in the methylerythritol phosphate(MEP) and/or the mevalonic acid (MVA) pathway to catalyze the conversionof glucose or other carbon sources to isopentenyl pyrophosphate (IPP)and/or dimethylallyl pyrophosphate (DMAPP). In some embodiments, themicrobial host cell further expresses an enzyme catalyzing theconversion of IPP and/or DMAPP to farnesyl diphosphate (FPP), allowingfor rotundone to be produced from sugar or other carbon sources (carbonsubstrates such as C1, C2, C3, C4, C5, and/or C6 carbon substrates). Insome embodiments, the host cell is a bacteria engineered to increasecarbon flux through the MEP pathway.

In some embodiments, the microbial host cell expresses an α-guaieneoxidase (αGOX) enzyme, which may be a P450 enzyme, non-heme ironoxygenase (NHIO), or laccase providing for biotransformation ofα-guaiene substrate. α-Guaiene substrate can be added to whole cell orcellular extracts or purified enzyme. In some embodiments, the cellfurther expresses at least one cytochrome P450 reductase to support P450enzyme activity for whole cell bioconversion processes. In someembodiments, the αGOX produces rotundone as the predominant oxygenatedproduct.

In some embodiments, the microbial host cell further expresses one ormore alcohol dehydrogenases. In some embodiments, the alcoholdehydrogenase converts one or more alcohol intermediates, produced bythe reaction of α-guaiene with αGOX, to rotundone.

In some embodiments, the microbial host cell is prokaryotic oreukaryotic, and may be a bacteria or yeast.

Other aspects and embodiments of the invention will be apparent from thefollowing detailed disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the chemical structure of rotundone.

FIG. 2 illustrates a biosynthetic pathway for the production ofrotundone. Farnesyl diphosphate is converted to α-guaiene by anα-guaiene terpene synthase (αGTPS) enzyme; and α-guaiene is converted to(−)-rotundone by an α-guaiene oxidase (αGOX) enzyme.

FIG. 3 illustrates that the production of (−)-rotundone from α-guaienecan proceed directly from the sesquiterpene precursor, or can involvethe production of one or both alcohol intermediates which aresubsequently converted to the ketone by an enzyme with alcoholdehydrogenase activity.

FIG. 4 shows results from the screening of amino acid substitutions inan exemplary α-guaiene terpene synthase (AcDGuaS3, SEQ ID NO: 8).Derivatives were tested for α-guaiene production in E. coli. The figureshows fold improvement in α-guaiene production.

FIG. 5 lists the altered profile toward production of α-guaiene in E.coli for several amino acid substitutions in AcDGuaS3. Fold improvementof α-guaiene as a % of the total products is listed.

FIG. 6 shows production of α-guaiene with expression of wild-typeAcDGuaS3 (α-GS) and mutant AcDGuaS3 having an F406L mutation (α-GS1) inE. coli.

FIGS. 7A and 7B show production of rotundol and rotundone in E. coliexpressing α-GS1 and an exemplary CYP450 system (engineered kaureneoxidase; KOeng). Expression of Vitis vinifera dehydrogenase (VvDH) alongwith α-GS1, KOeng as the α-guaiene oxidase, and Camptotheca acuminatacytochrome P450 reductase (CaCPR) reduces the titer of rotundol (FIG.7A) and increases rotundone titer (FIG. 7B).

FIGS. 8A and B show Gas-Chromatography/Mass Spectrometry (GC/MS)confirmation of production of rotundone from E. coli strain expressingα-GS1, KOeng, CaCPR, and VvDH. FIG. 8A shows the abundance of rotundonein GC/MS and FIG. 8B shows the gas chromatogram for rotundone.

FIG. 9 shows production of rotundol and rotundone in E. coli expressingα-GS1 and an alternative CYP450 system. Expression of α-GS1, Helianthusannuus germacrene A monooxygenase engineered for expression in E. coli(HaGAO), and AaCPR (Artemisia annua cytochrome P450 reductase) producesprimarily rotundone.

FIG. 10 shows a multiple sequence alignment of five kaurene oxygenases,including KOeng (SEQ ID NO: 51), which functions as α-guaiene oxidaseenzyme. The homologs are HaKO (SEQ ID NO: 50), AaKO (SEQ ID NO: 49),CcKO (SEQ ID NO: 47), LsKO (SEQ ID NO: 46), and NtKO (SEQ ID NO: 45).

DETAILED DESCRIPTION

In various aspects, the present disclosure provides microbial host cells(or microbial host strains) and methods for producing rotundone andmethods of making products containing rotundone, such as flavor andfragrance products, among others. In other aspects, the presentinvention provides enzymes and polynucleotides encoding enzymes for theproduction of rotundone.

In some embodiments, the present disclosure provides a microbial hostcell, including bacteria and yeast, expressing an enzyme pathwaycatalyzing the conversion of farnesyl diphosphate (FPP) to rotundone. Invarious embodiments, the enzymatic pathway comprises a α-guaienesynthase enzyme (αGTPS) and an α-guaiene oxidase (αGOX) enzyme. In theseembodiments, the microbial cells can synthesize rotundone product fromany suitable carbon source. In some embodiments, the specificity of theα-guaiene synthase enzyme enables production of rotundone at high yieldwith fewer terpenoid byproducts. In some embodiments, the microbial hostcell may further expresses one or more alcohol dehydrogenase enzymes(ADH). In some embodiments, the ADH converts one or more alcoholintermediates, produced by the reaction of α-guaiene with αGOX, torotundone.

In some embodiments, the microbial host cell expresses an α-guaieneoxidase (αGOX) enzyme, providing for biotransformation of α-guaienesubstrate. In some embodiments, αGOX is a P450 enzyme, non-heme ironoxygenase (NHIO), or laccase. In some embodiments, the cell may furtherexpress a cytochrome P450 reductase to support P450 activity. In someembodiments, the microbial host cell may further express one or morealcohol dehydrogenase enzymes (ADH). In some embodiments, the ADHconverts one or more alcohol intermediates, produced by the reaction ofα-guaiene with αGOX, to rotundone.

Rotundone comprises a guaiene carbon skeleton with a single ketone groupin the carbon 2 position (see FIG. 1). α-Guaiene is the precursor torotundone. α-Guaiene is a sesquiterpene hydrocarbon found in oilextracts from various plants. While α-guaiene can be converted torotundone by aerial oxidation or enzymatic transformation, theseprocesses are not efficient, in part due to the specificity of enzymesused.

A biosynthetic pathway for rotundone is shown in FIG. 2. The C15sesquiterpene precursor substrate farnesyl diphosphate (FPP) is cyclizedto α-guaiene by an αGTPS terpene synthase enzyme. The α-guaiene (i.e.,cyclized FPP) is then oxidized to rotundone via an αGOX enzyme. Theproduction of the ketone moiety in α-guaiene resulting in rotundone canproceed directly, or can alternatively proceed through alcoholintermediates, with either stereochemistry of the alcohol intermediate,i.e., (2R)-rotundol or (2S)-rotundol (see FIG. 3).

The αGTPS enzyme is a terpene synthase enzyme (TPS). TPS enzymes areresponsible for the synthesis of the terpene molecules from two isomeric5-carbon precursor building blocks, leading to 5-carbon isoprene,10-carbon monoterpenes, 15-carbon sesquiterpenes and 20-carbonditerpenes. The structures and functions of TPS enzymes are described inChen et al., The Plant Journal, 66: 212-229 (2011). Tobacco5-epi-aristolochene synthase, a terpene synthase, has been describedalong with structural coordinates, including key active sitecoordinates. These structural coordinates can be used for constructinghomology models of αGTPS enzymes, which are useful for guiding theengineering of αGTPS enzymes with improved specificity and productivity.See, U.S. Pat. Nos. 6,645,762, 6,495,354, and 6,645,762, which arehereby incorporated by reference in their entireties.

In some embodiments, the TPS enzyme is selected from Vitis vinifera GuaS(VvGuas) enzyme (SEQ ID NO: 1), patchouli synthase (PcPS) enzyme fromPogostemon cablin (Uniprot Q49SP3) (SEQ ID NO: 2), Vitis viniferagermacrene D synthase (VvGDS; NCBI Ref #XP_002282488.1) (SEQ ID NO: 21),or a variant thereof. In some embodiments, the TPS enzyme is selectedfrom Aquilaria crassna, for example, AcC1 (Uniprot DOVMR5); AcC2(Uniprot DOVMR6) (SEQ ID NO: 3); AcC3 (Uniprot DOVMR7) (SEQ ID NO: 4);or AcC4 (Uniprot DOVMR8) (SEQ ID NO: 5), or a variant thereof. In someembodiments, the A. crassna TPS is a mutant of AcC1, for exampleAcC1mut1-M42 (SEQ ID NO: 6) and AcClmut2-M50 (SEQ ID NO: 7). Other TPSenzymes are provided herein as SEQ ID NO:8 (Aquilaria crassna AcDGuaS3),SEQ ID NO:9 (Aquilaria crassna AcDGuaS4), SEQ ID NO:10 (Aquilariacrassna AcDGuaS2), SEQ ID NO:11 (Aquilaria crassna AcDGuaS5), SEQ ID NO:12 (Aquilaria spp. AmiDGuaS1), SEQ ID NO: 13 (Aquilaria spp. AmiDGuaS2),SEQ ID NO: 14 (Aquilaria spp. AmiDGuaS3), SEQ ID NO: 15 (Aquilaria spp.AmaDGuaS1), SEQ ID NO: 16 (Aquilaria spp. AmaDGuaS2), SEQ ID NO: 17(Aquilaria spp. AsDGuaS1), SEQ ID NO: 18 (Aquilaria spp. AsDGuaS2), SEQID NO: 19 (Aquilaria spp. AsDGuaS3), and SEQ ID NO: 20 (Aquilaria spp.AsDGuaS4), or a variant thereof.

Terpene synthase variants include α-guaiene synthase enzymes comprisingan amino acid sequence that has 50% or more sequence identity with anyone of SEQ ID NOs: 1 to 21. In some embodiments, the variant comprisesan amino acid sequence that has at least about 60% sequence identity, orat least about 70% sequence identity, or at least about 80% sequenceidentity, or at least about 90% sequence identity, or at least about 95%sequence identity, or at least about 98% sequence identity, or at leastabout 99% sequence identity with the amino acid sequence of any one SEQID NOs: 1 to 21. In some embodiments, the variant includes from 1 toabout 20, or from 1 to about 10, or from 1 to about 5 amino acidmodifications independently selected from substitutions, deletions, andinsertions to an amino acid sequence selected from SEQ ID NOs: 1 to 21.In some embodiments, the terpene synthase comprises a substitution toone or more of the substrate binding site or active site. In someembodiments, modifications to enzymes can be informed by construction ofa homology model. In some embodiments, the amino acid modifications canbe selected to improve one or more of: enzyme productivity, selectivityfor the desired substrate and/or product, stability, temperaturetolerance, and expression.

In some embodiments, the α-guaiene synthase enzyme comprises an aminoacid sequence that has at least 50% sequence identity with any one ofSEQ ID NOs: 1, 3, 4, 6 to 10, 11 to 15, or 19. In some embodiments, theα-guaiene synthase enzyme comprises an amino acid sequence that has atleast about 60% sequence identity, or at least about 70% sequenceidentity, or at least about 80% sequence identity, or at least about 90%sequence identity, or at least about 95% sequence identity, or at leastabout 98% sequence identity, or at least about 99% sequence identitywith the amino acid sequence of any one SEQ ID NOs: 1, 3, 4, 6 to 10, 11to 15, or 19. In some embodiments, the α-guaiene synthase enzymeincludes from 1 to about 20, or from 1 to about 10, or from 1 to about 5amino acid modifications independently selected from substitutions,deletions, and insertions to an amino acid sequence selected from SEQ IDNOs: 1, 3, 4, 6 to 10, 11 to 15, or 19.

In some embodiments, the α-guaiene synthase enzyme comprises an aminoacid sequence that has 50% or more sequence identity with SEQ ID NO: 8.In some embodiments, the α-guaiene synthase enzyme comprises an aminoacid sequence that has at least about 60% sequence identity, or at leastabout 70% sequence identity, or at least about 80% sequence identity, orat least about 90% sequence identity, or at least about 95% sequenceidentity, or at least about 98% sequence identity, or at least about 99%sequence identity with the amino acid sequence of SEQ ID NO: 8. In someembodiments, the α-guaiene synthase enzyme includes from 1 to about 20,or from 1 to about 10, or from 1 to about 5 amino acid modificationsindependently selected from substitutions, deletions, and insertions tothe amino acid sequence of SEQ ID NO: 8.

In some embodiments, the α-guaiene synthase may have one, two, three,four, five or more amino acid substitutions at positions correspondingto positions selected from 72, 273, 290, 368, 371, 374, 377, 381, 382,399, 406, 419, 433, 442, 443, 454, 512, and 522 of SEQ ID NO: 8. Forexample, in some embodiments the α-GTPS comprises an amino acid sequencehaving one or more (e.g, 2, 3, 4, 5, or all) of the amino acidsubstitutions selected from T72I, M273L, R290K, F368M, I371L, S374A,R377V, Y381W, F382L, I399V, F406L, L419T, V433I, Y442L, I443M, E454K,F512L, and K522D relative to SEQ ID NO: 8. In some embodiments, theα-GTPS includes an amino acid substitution at the position correspondingto position 406 of SEQ ID NO: 8, and which is optionally F406L, F406A,F4061, F406V, or F406G. In some embodiments, the α-GTPS enzyme includesan amino acid substitution at the position corresponding to position 443of SEQ ID NO: 8, which is optionally I443M. In some embodiments, theα-GTPS enzyme includes a mutation at the position corresponding toposition 512 of SEQ ID NO: 8, which is optionally F512L, F512A, F512I,F512V, or F512G.

Amino acid substitutions may be conservative or non-conservativesubstitutions.

“Conservative substitutions” may be made, for instance, on the basis ofsimilarity in polarity, charge, size, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the amino acid residuesinvolved. The 20 naturally occurring amino acids can be grouped into thefollowing six standard amino acid groups:

(1) hydrophobic: Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro; and

(6) aromatic: Trp, Tyr, Phe.

As used herein, “conservative substitutions” are defined as exchanges ofan amino acid by another amino acid listed within the same group of thesix standard amino acid groups shown above. For example, the exchange ofAsp by Glu retains one negative charge in the so modified polypeptide.In addition, glycine and proline may be substituted for one anotherbased on their ability to disrupt α-helices. Some preferred conservativesubstitutions within the above six groups are exchanges within thefollowing sub-groups: (i) Ala, Val, Leu and Ile; (ii) Ser and Thr; (ii)Asn and Gln; (iv) Lys and Arg; and (v) Tyr and Phe.

As used herein, “non-conservative substitutions” or “non-conservativeamino acid exchanges” are defined as exchanges of an amino acid byanother amino acid listed in a different group of the six standard aminoacid groups (1) to (6) shown above.

Mutations in α-GTPS enzymes can be guided by homology models usingmolecular structures/models of sesquiterpene synthase disclosed in Drewet al., J. of Exp. Botany, Vol. 67, No. 3, pp. 799-808 (2015) and/orKumeta et al., Plant Physiology, Vol. 154, pp. 1998-2007 (2010), whichare hereby incorporated by reference in its entirety.

The similarity of nucleotide and amino acid sequences, i.e. thepercentage of sequence identity, can be determined via sequencealignments. Such alignments can be carried out with several art-knownalgorithms, such as with the mathematical algorithm of Karlin andAltschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) orwith the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T.J. (1994) Nucleic Acids Res. 22, 4673-80). The grade of sequenceidentity (sequence matching) may be calculated using e.g. BLAST, BLAT orBlastZ (or BlastX). A similar algorithm is incorporated into the BLASTNand BLASTP programs of Altschul et al (1990) J. Mol. Biol. 215: 403-410.BLAST protein searches may be performed with the BLASTP program,score=50, word length=3. To obtain gapped alignments for comparativepurposes, Gapped BLAST is utilized as described in Altschul et al (1997)Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs are used.Sequence matching analysis may be supplemented by established homologymapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b,19 Suppl 1:154-162) or Markov random fields.

TPS enzymes can generate multiple products with the guaiene skeletonfrom FPP with varied amounts of α-guaiene produced by different TPSenzymes. In some embodiments, the α-guaiene synthase (or engineeredvariant) produces predominantly α-guaiene (e.g., greater than 50%) asthe product from FPP substrate. In some embodiments, the α-guaienesynthase produces greater than about 75%, or greater than about 80%, orgreater than about 85%, or greater than about 90%, or greater than about95% α-guaiene as the product from FPP. Enzyme specificity can bedetermined in host microbial cells producing FPP and expressing theα-guaiene synthase, followed by chemical analysis of total terpenoidproducts. In some embodiments, the α-guaiene produced in the αGTPSreaction is oxidized to rotundone. In some embodiments, an αGOX enzymeoxidizes α-guaiene to rotundone. In some embodiments, the αGOX oxidizesat least one portion of the α-guaiene to a ketone. In some embodiments,the oxidation of α-guaiene by αGOX results in the production of one ormore alcohol intermediates. In some embodiments, the alcoholintermediates are converted to rotundone by one or more alcoholdehydrogenases.

In some embodiments, the αGOX enzyme is a cytochrome P450 (CYP450)enzyme. CYP450 enzymes are involved in the formation (synthesis) andbreakdown (metabolism) of various molecules and chemicals within cells.CYP450 enzymes have been identified in all kingdoms of life (i.e.,animals, plants, fungi, protists, bacteria, archaea, and even inviruses). Illustrative structure and function of CYP450 enzymes aredescribed in Uracher et al., TRENDS in Biotechnology, 24(7): 324-330(2006). In some embodiments, the P450 enzymes are engineered to have adeletion of all or part of the wild type N-terminal transmembraneregion, and the addition of a transmembrane domain derived from an E.coli inner membrane cytoplasmic C-terminus protein. In variousembodiments, the transmembrane domain is a single-pass transmembranedomain. In various embodiments, the transmembrane domain (or “N-terminalanchor”) is derived from an E. coli gene selected from waaA, ypfN, yhcB,yhbM, yhhm, zipA, ycgG, djlA, sohB, lpxK, F11O, motA, htpx, pgaC, ygdD,hemr, and ycls. These genes were identified as inner membranecytoplasmic C-terminus proteins through bioinformatic prediction as wellas experimental validation. The invention may employ an N-terminalanchor sequence that is a derivative of the E. coli wild-typetransmembrane domain, that is, having one or more mutations (e.g., aminoacid substitutions) with respect to the wild-type sequence. Methods ofmaking such engineered P450 enzymes as well as engineered P450 enzymesare described in U.S. Patent Publication No. 2018/0251738, which ishereby incorporated by reference in its entirety.

In some embodiments, the CYP450 is selected from the V. vinifera VvSTO2(CYP71BE5; Uniprot F6I534) (SEQ ID NO: 22); Bacillus subtilis CYP152A1(Uniprot 031440) (SEQ ID NO: 23); B. subtilis CYP107K1 (Uniprot A5HNX5)(SEQ ID NO: 24); Bacillus cereus CYP106 (Uniprot Q737I9) (SEQ ID NO:25); and B. cereus CYP107 (Uniprot Q737F2) (SEQ ID NO: 26); or a variantthereof.

αGOX variants include enzymes comprising an amino acid sequence that has50% or more sequence identity with any one of SEQ ID NOS: 22 to 26. Insome embodiments, the variant comprises an amino acid sequence that hasat least about 60% sequence identity, or at least about 70% sequenceidentity, or at least about 80% sequence identity, or at least about 90%sequence identity, or at least about 95% sequence identity, or at leastabout 98% sequence identity, or at least about 99% sequence identitywith the amino acid sequence of any one SEQ ID NOS: 22 to 26. In someembodiments, the variant includes from 1 to about 20, or from 1 to about10, or from 1 to about 5 amino acid modifications independently selectedfrom substitutions, deletions, and insertions to an amino acid sequenceselected from SEQ ID NO: 22 to 26. In some embodiments, the oxygenasecomprises a substitution to one or more of the substrate binding site oractive site. In some embodiments, modifications to enzymes can beinformed by construction of a homology model. In some embodiments,selection and modification of enzymes is informed by assaying activityon α-guaiene substrate. In some embodiments, the amino acidmodifications can be selected to improve one or more of: enzymeproductivity, selectivity for the desired substrate and/or product,stability, temperature tolerance, and expression.

In some embodiments, the αGOX enzyme is a non-heme iron oxygenase (NHIO)or a laccase. In some embodiments, the laccase is derived from bacteriaor fungi (including filamentous fungi and yeasts). By way of example, insome embodiments, the laccase is from a species selected fromAspergillus, Neurospora (e.g., N. crassa), Podospora, Botrytis,Collybia, Fomes, Lentinus, Lentinus, Pleurotus, Trametes, Rhizoctonia(e.g., R. solani), Coprinus (e.g., C. plicatilis), Psatyrella,Mycehophtera (e.g., M thermophila), Schytalidium, and Polyporus, (e.g.,P. pinsitus), Phiebia, and Coriolus, or is a derivative thereof.

In some embodiments, the CYP450 (αGOX) comprises an amino acid sequencethat has at least 50% identity to SEQ ID NO: 51. In some embodiments,the CYP450 enzyme comprises an amino acid sequence that is at leastabout 55%, or at least about 60%, or at least about 65%, or at leastabout 70%, or at least about 75%, or at least about 80%, or at leastabout 85%, or at least about 90%, or at least about 95%, or at least98%, or at least 99% identical to SEQ ID NO: 51. For example, the CYP450enzyme may comprise an amino acid sequence having from 1 to 20 or from 1to 10 amino acid modifications with respect to SEQ ID NO: 51, the aminoacid modifications being independently selected from amino acidsubstitutions, deletions, and insertions with respect to correspondingpositions in SEQ ID NO: 51.

In some embodiments, the CYP450 comprises an amino acid sequence thathas at least 50% identity to SEQ ID NO: 52. In some embodiments, theCYP450 enzyme comprises an amino acid sequence that is at least about55%, or at least about 60%, or at least about 65%, or at least about70%, or at least about 75%, or at least about 80%, or at least about85%, or at least about 90%, or at least about 95%, or at least 98%, orat least 99% identical to SEQ ID NO: 52. For example, the CYP450 enzymemay comprise an amino acid sequence having from 1 to 20 or from 1 to 10amino acid modifications with respect to SEQ ID NO: 52, the amino acidmodifications being independently selected from amino acidsubstitutions, deletions, and insertions with respect to correspondingpositions in SEQ ID NO: 52.

In some embodiments, the CYP450 comprises an amino acid sequence thathas at least 50% identity to SEQ ID NOs: 54, 55, or 56. In someembodiments, the CYP450 enzyme comprises an amino acid sequence that isat least about 55%, or at least about 60%, or at least about 65%, or atleast about 70%, or at least about 75%, or at least about 80%, or atleast about 85%, or at least about 90%, or at least about 95%, or atleast 98%, or at least 99% identical to SEQ ID NOs: 54, 55, or 56. Forexample, the CYP450 enzyme may comprise an amino acid sequence havingfrom 1 to 20 or from 1 to 10 amino acid modifications with respect toSEQ ID NO: 54, 55, or 56, the amino acid modifications beingindependently selected from amino acid substitutions, deletions, andinsertions with respect to corresponding positions in SEQ ID NO: 54, 55,or 56.

Amino acid modification to CYP450 enzymes can be guided by availablestructures, including those described in Pallan et al., “Structural andkinetic basis of steroid 17a, 20-lyase activity in teleost fishcytochrome P450 17A1 and its absence in cytochrome P450 17A2,” Journalof Biological Chemistry 290.6 (2015): 3248-3268, which is herebyincorporated by reference in its entirety. Pallan et al. describe aZebra fish cytochrome P450 17A2 along with structural coordinates,including key active site coordinates. These structural coordinates canbe used for constructing homology models of CYP450 enzymes, which areuseful for guiding the engineering of CYP450 enzymes with improvedspecificity and productivity.

In some embodiments, the CYP450 enzyme requires the presence of anelectron transfer protein capable of transferring electrons to theCYP450 protein. In some embodiments, this electron transfer protein is acytochrome P450 reductase (CPR), which can be expressed by the microbialhost cell. Various reductases that may be used are described in U.S.Patent Publication No. 2018/0135081, which is hereby incorporated byreference in its entirety.

Exemplary P450 reductase enzymes include those shown herein as SEQ IDNOs: 27 to 34 or 53, or a variant thereof. Variants generally includeenzymes comprising an amino acid sequence that has 50% or more sequenceidentity with any one of SEQ ID NOs: 27 to 34 or 53. In someembodiments, the variant comprises an amino acid sequence that has atleast about 60% sequence identity, or at least about 70% sequenceidentity, or at least about 80% sequence identity, or at least about 90%sequence identity, or at least about 95% sequence identity, or at leastabout 98% sequence identity, or at least about 99% sequence identitywith the amino acid sequence of any one SEQ ID NOs: 27 to 34 or 53. Insome embodiments, the variant includes from 1 to about 20, or from 1 toabout 10, or from 1 to about 5 amino acid modifications independentlyselected from substitutions, deletions, and insertions to an amino acidsequence selected from SEQ ID NOs: 27 to 34 or 53.

In some embodiments, the CPR comprises an amino acid sequence that hasat least 50% identity to SEQ ID NO: 53 (CaCPR). In some embodiments, theCPR enzyme comprises an amino acid sequence that is at least about 55%,or at least about 60%, or at least about 65%, or at least about 70%, orat least about 75%, or at least about 80%, or at least about 85%, or atleast about 90%, or at least about 95%, or at least 98%, or at least 99%identical to SEQ ID NO: 53. For example, the CPR enzyme may comprise anamino acid sequence having from 1 to 20 or from 1 to 10 amino acidmodifications with respect to SEQ ID NO: 53, the amino acidmodifications being independently selected from amino acidsubstitutions, deletions, and insertions with respect to correspondingpositions in SEQ ID NO: 53.

In some embodiments, the αGOX reaction results in hydroxylation ofα-guaiene, thereby producing one or more alcohol intermediates, e.g.,(2R)-rotundol or (2S)-rotundol (see FIG. 3). In some embodiments, theαGOX further oxidizes at least a portion of the α-guaiene to a ketone.In some embodiments, the alcohol intermediates (e.g., (2R)-rotundol or(2S)-rotundol) are converted to rotundone by one or more alcoholdehydrogenases (ADHs). Thus, in some embodiments, the microbial hostcell expresses one or more alcohol dehydrogenases (ADH). By way ofexample, in some embodiments, the ADH is selected from an enzymecomprising an amino acid sequence selected from SEQ ID NOs: 35 to 44, ora variant thereof. Variants generally include enzymes comprising anamino acid sequence that has 50% or more sequence identity with any oneof SEQ ID NOs: 35 to 44. In some embodiments, the variant comprises anamino acid sequence that has at least about 60% sequence identity, or atleast about 70% sequence identity, or at least about 80% sequenceidentity, or at least about 90% sequence identity, or at least about 95%sequence identity, or at least about 98% sequence identity, or at leastabout 99% sequence identity with the amino acid sequence of any one SEQID NOS: 35 to 44. In some embodiments, the variant includes from 1 toabout 20, or from 1 to about 10, or from 1 to about 5 amino acidmodifications independently selected from substitutions, deletions, andinsertions to an amino acid sequence selected from SEQ ID NO: 35 to 44.In some embodiments, the amino acid modifications can be selected toimprove one or more of: enzyme productivity, selectivity for the desiredsubstrate and/or product, stability, temperature tolerance, andexpression.

In some embodiments, the ADH comprises an amino acid sequence that hasat least 50% identity to SEQ ID NO: 43 (VvDH). In some embodiments, theADH enzyme comprises an amino acid sequence that is at least about 55%,or at least about 60%, or at least about 65%, or at least about 70%, orat least about 75%, or at least about 80%, or at least about 85%, or atleast about 90%, or at least about 95%, or at least 98%, or at least 99%identical to SEQ ID NO: 43. For example, the ADH enzyme may comprise anamino acid sequence having from 1 to 20 or from 1 to 10 amino acidmodifications with respect to SEQ ID NO: 43, the amino acidmodifications being independently selected from amino acidsubstitutions, deletions, and insertions with respect to correspondingpositions in SEQ ID NO: 43.

In some embodiments, the microbial cell expresses an αGOX, and producespredominately rotundone (e.g., at least 75% of the oxygenated product isrotundone), without expression of an ADH enzyme.

In various embodiments, the αGTPS and αGOX are expressed together in anoperon, or are expressed individually. The enzymes may be expressed fromextrachromosomal elements such as plasmids, or bacterial artificialchromosomes, or may be chromosomally integrated.

In some embodiments, the cell does not express an αGTPS, but expressesan α-guaiene oxidase (αGOX), allowing enzymatic biotransformation ofα-guaiene, which can take place with whole cells or whole or partiallypurified extracts of cells.

In some embodiments, the αGOX and/or the ADH are provided in a purifiedrecombinant form for production of rotundone from α-guaiene, or(2R)-rotundol or (2S)-rotundol, in a cell free system.

In some embodiments, the microbial host cell is also engineered toexpress or overexpress one or more enzymes in the methylerythritolphosphate (MEP) and/or the mevalonic acid (MVA) pathway to catalyzeisopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP)from glucose or other carbon source.

In some embodiments, the microbial host cell is engineered to express oroverexpress one or more enzymes of the MEP pathway. In some embodiments,the MEP pathway is increased and balanced with downstream pathways byproviding duplicate copies of certain rate-limiting enzymes. The MEP(2-C-methyl-D-erythritol 4-phosphate) pathway, also called the MEP/DOXP(2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate)pathway or the non-mevalonate pathway or the mevalonic acid-independentpathway refers to the pathway that converts glyceraldehyde-3-phosphateand pyruvate to IPP and DMAPP. The pathway typically involves action ofthe following enzymes: 1-deoxy-D-xylulose-5-phosphate synthase (Dxs),1-deoxy-D-xylulose-5-phosphate reductoisomerase (IspC),4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD),4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE),2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF),1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG), andisopentenyl diphosphate isomerase (IspH). The MEP pathway, and the genesand enzymes that make up the MEP pathway, are described in U.S. Pat. No.8,512,988, which is hereby incorporated by reference in its entirety.For example, genes that make up the MEP pathway include dxs, ispC, ispD,ispE, ispF, ispG, ispH, idi, and ispA. In some embodiments, themicrobial host cell expresses or overexpresses of one or more of dxs,ispC, ispD, ispE, ispF, ispG, ispH, idi, ispA, or modified variantsthereof, which results in the increased production of IPP and DMAPP. Insome embodiments, rotundone is produced at least in part by metabolicflux through an MEP pathway, and wherein the microbial host cell has atleast one additional gene copy of one or more of dxs, ispC, ispD, ispE,ispF, ispG, ispH, idi, ispA, or modified variants thereof.

In some embodiments, the microbial host cell is engineered to express oroverexpress one or more enzymes of the MVA pathway. The MVA pathwayrefers to the biosynthetic pathway that converts acetyl-CoA to IPP. Themevalonate pathway typically comprises enzymes that catalyze thefollowing steps: (a) condensing two molecules of acetyl-CoA toacetoacetyl-CoA (e.g., by action of acetoacetyl-CoA thiolase); (b)condensing acetoacetyl-CoA with acetyl-CoA to formhydroxymethylglutaryl-CoenzymeA (HMG-CoA) (e.g., by action of HMG-CoAsynthase (HMGS)); (c) converting HMG-CoA to mevalonate (e.g., by actionof HMG-CoA reductase (HMGR)); (d) phosphorylating mevalonate tomevalonate 5-phosphate (e.g., by action of mevalonate kinase (MK)); (e)converting mevalonate 5-phosphate to mevalonate 5-pyrophosphate (e.g.,by action of phosphomevalonate kinase (PMK)); and (f) convertingmevalonate 5-pyrophosphate to isopentenyl pyrophosphate (e.g., by actionof mevalonate pyrophosphate decarboxylase (MPD)). The MVA pathway, andthe genes and enzymes that make up the MVA pathway, are described inU.S. Pat. No. 7,667,017, which is hereby incorporated by reference inits entirety. In some embodiments, the microbial host cell expresses oroverexpresses one or more of acetoacetyl-CoA thiolase, HMGS, HMGR, MK,PMK, and MPD or modified variants thereof, which results in theincreased production of IPP and DMAPP. In some embodiments, rotundone isproduced at least in part by metabolic flux through an MVA pathway, andwherein the microbial host cell has at least one additional gene copy ofone or more of acetoacetyl-CoA thiolase, HMGS, HMGR, MK, PMK, MPD, ormodified variants thereof.

In some embodiments, the microbial host cell is engineered to increaseproduction of IPP and DMAPP from glucose as described in PCT ApplicationNos. PCT/US2018/016848 and PCT/US2018/015527, the contents of which arehereby incorporated by reference in their entireties. For example, insome embodiments the microbial host cell overexpresses MEP pathwayenzymes, with balanced expression to push/pull carbon flux to IPP andDMAPP. In some embodiments, the microbial host cell is engineered toincrease the availability or activity of Fe—S cluster proteins, so as tosupport higher activity of IspG and IspH, which are Fe—S enzymes. Insome embodiments, the host cell is engineered to overexpress IspG andIspH, so as to provide increased carbon flux to1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP) intermediate, butwith balanced expression to prevent accumulation of HMBPP at an amountthat reduces cell growth or viability, or at an amount that inhibits MEPpathway flux.

Conversion of IPP and DMAPP precursors to farnesyl diphosphate (FPP) istypically through the action of a farnesyl diphosphate synthase (FPPS).Exemplary FPPS enzymes are disclosed in US 2018/0135081, which is herebyincorporated by reference in its entirety.

In some embodiments, the host cell is engineered to downregulate theubiquinone biosynthesis pathway, e.g., by reducing the expression oractivity of IspB, which uses IPP and FPP substrate.

In some embodiments, the microbial host cell is a bacteria selected fromEscherichia spp., Bacillus spp., Corynebacterium spp., Rhodobacter spp.,Zymomonas spp., Vibrio spp., and Pseudomonas spp. For example, in someembodiments, the bacterial host cell is a species selected fromEscherichia coli, Bacillus subtilis, Corynebacterium glutamicum,Rhodobacter capsulatus, Rhodobacter sphaeroides, Zymomonas mobilis,Vibrio natriegens, or Pseudomonas putida. In some embodiments, thebacterial host cell is E. coli.

In some embodiments, the microbial host cell is a species ofSaccharomyces, Pichia, or Yarrowia, including, but not limited to,Saccharomyces cerevisiae, Pichia pastoris, and Yarrowia lipolytica.

In another aspect, the present disclosure provides a method for makingrotundone. The method comprises providing a microbial host cell (ormicrobial host strain) as disclosed herein. The microbial host cellexpresses an αGOX enzyme, and optionally an αGTPS enzyme as describedherein. Cells expressing an αGOX enzyme can be used for bioconversion ofα-guaiene using whole cells or cell extracts. Cells expressing an αGOXenzyme and an αGTPS enzyme can produce rotundone from a carbon source.

In some embodiments, the microbial host cell further expresses one ormore alcohol dehydrogenases (ADHs) disclosed herein. Cells expressingADHs can convert alcohol intermediates produced by the αGOX reactioninto rotundone.

In some embodiments, the host cell is cultured to produce rotundone. Insome embodiments, microbial cells are cultured with carbon substrates(sources) such as C1, C2, C3, C4, C5, and/or C6 carbon substrates. Inexemplary embodiments, the carbon source is glucose, sucrose, fructose,xylose, and/or glycerol. Culture conditions are generally selected fromaerobic, microaerobic, and anerobic.

In various embodiments, the host cell is cultured at a temperaturebetween 22° C. and 37° C. While commercial biosynthesis in bacteria suchas E. coli can be limited by the temperature at which overexpressedand/or foreign enzymes (e.g., enzymes derived from plants) are stable,recombinant enzymes (including the terpenoid synthase) may be engineeredto allow for cultures to be maintained at higher temperatures, resultingin higher yields and higher overall productivity. In some embodiments,the host cell is a bacterial host cell, and culturing is conducted atabout 22° C. or greater, about 23° C. or greater, about 24° C. orgreater, about 25° C. or greater, about 26° C. or greater, about 27° C.or greater, about 28° C. or greater, about 29° C. or greater, about 30°C. or greater, about 31° C. or greater, about 32° C. or greater, about33° C. or greater, about 34° C. or greater, about 35° C. or greater,about 36° C. or greater, or about 37° C.

Rotundone can be extracted from media and/or whole cells, and recovered.In some embodiments, the oxygenated rotundone product is recovered andoptionally enriched by fractionation (e.g. fractional distillation). Theoxygenated product can be recovered by any suitable process, includingpartitioning the desired product into an organic phase. The productionof the desired product can be determined and/or quantified, for example,by gas chromatography (e.g., GC-MS). The desired product can be producedin batch or continuous bioreactor systems. Production of product,recovery, and/or analysis of the product can be done as described in US2012/0246767, which is hereby incorporated by reference in its entirety.For example, in some embodiments, oxidized oil is extracted from aqueousreaction medium, which may be done by partitioning into an organicphase, followed by fractional distillation. Sesquiterpene andsesquiterpenoid components of fractions may be measured quantitativelyby GC/MS, followed by blending of the fractions.

In some embodiments, the microbial host cells and methods disclosedherein are suitable for commercial production of rotundone, that is, themicrobial host cells and methods are productive at commercial scale. Insome embodiments, the size of the culture is at least about 100 L, atleast about 200 L, at least about 500 L, at least about 1,000 L, atleast about 10,000 L, at least about 100,000 L, or at least about1,000,000 L. In some embodiment, the culturing may be conducted in batchculture, continuous culture, or semi-continuous culture.

In some aspects, the present disclosure provides methods for making aproduct comprising rotundone, including flavor and fragrancecompositions or products. In some embodiments, the method comprisesproducing rotundone as described herein through microbial culture,recovering the rotundone, and incorporating the rotundone into theflavor or fragrance composition, or a consumable product (e.g., a foodproduct).

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a cell” includesa combination of two or more cells, and the like.

As used herein, the term “about” in reference to a number is generallytaken to include numbers that fall within a range of 10% in eitherdirection (greater than or less than) of the number.

EXAMPLES

Rotundone is a bicyclic sesquiterpene (FIG. 1) and is responsible forpepper aromas in grapes and wine and in herbs and spices, especiallyblack and white pepper, where it has a high odor activity value (OAV).The biosynthesis of rotundone involves cyclization of the C15sesquiterpene precursor substrate farnesyl diphosphate (FPP) toα-guaiene by an α-GTPS terpene synthase (FIG. 2). Enzymatic oxygenationof α-guaiene can produce rotundone, and may proceed through an alcoholintermediate (FIGS. 2 and 3). For example, α-guaiene may be converted to(2S)-rotundol or (2R)-rotundol by the action of αGOX, and the alcoholintermediate(s) (rotundol) can be converted to rotundone by the actionof the αGOX or an alcohol dehydrogenase.

Example 1: Engineering α-Guaiene Synthase to Improve α-GuaieneProduction

The α-guaiene precursor, rotundol, or rotundone can be produced bybiosynthetic fermentation processes, using microbial strains thatproduce high levels of MEP pathway products, along with heterologousexpression of rotundone biosynthesis enzymes, including, enzymes thatcatalyze 1) cyclization of FPP to α-guaiene; 2) oxidation of α-guaieneto rotundone, which can include 3) dehydrogenation of rotundol torotundone. For example, in bacteria such as E. coli, isopentenylpyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) can beproduced from glucose or other carbon source, and which can be convertedto farnesyl diphosphate (FPP) by recombinant farnesyl diphosphatesynthase (FPPS). FPP is converted to α-guaiene by α-guaiene synthase(αGTPS) by cyclization. The α-guaiene is converted to rotundol orrotundone by oxygenation reaction catalyzed by an α-guaiene oxidase(αGOX). In instances where the αGOX enzyme catalyzes the production of(2S)-rotundol or (2R)-rotundol from α-guaiene, the conversion ofrotundol to rotundone may be catalyzed by a dehydrogenase.

Using an E. coli background strain that produces high levels of the MEPpathway products IPP and DMAPP (see US 2018/0245103 and US 2018/0216137,which are hereby incorporated by reference), candidate αGTPS enzymeswere screened by co-expression with FPPS. Fermentation was performed in96 well plates for 48 hours. The following synthase enzymes demonstratedproduction of α-guaiene in E. coli: AcC1mut1_M42, AcC1mut2 M50, AcC2,AcC3, AcDGuaS2, AcDGuaS3, AcDGuaS4, AcDGuaS5, AmaDGuaS1, AmiDGuaS1,AmiDGuaS2, AmiDGuaS3, AsDGuaS3, PcPS, and VvGuaS. In addition to thedesired α-guaiene product, active enzymes had varying product profiles.For example, all active Aquilaria enzymes showed α-bulnesene as a majorproduct with α-guaiene. VvGuaS accumulated α-bulnesene and globulol insimilar levels to α-guaiene. AcDGuaS3 was selected for subsequentstudies based on its productivity and product profile.

A panel of amino acid substitutions to the AcDGuaS3 sequence werescreened for their ability to convert FPP to α-guaiene in E. coli. Thefermentation was conducted in 96 well plates for 48 hours. FIG. 4 showsseveral mutants (i.e., amino acid substitutions) and the associatedfold-improvement in α-guaiene production. For example, F406Lsubstitution in AcDGuaS3 demonstrated a significantly improved titer ofα-guaiene (1.71 fold higher than wild-type). Amino acid substitutionswere further evaluated for substitutions that shift the product profiletoward α-guaiene. See FIG. 5. As shown, a single substitution inwild-type AcDGuaS3 (I443M) shows a 2.4 fold improvement in % α-guaienerelative to other products. Similarly, a F406L substitution shows a 2.12fold improvement in % α-guaiene relative to other products. A F512mutation demonstrated a 1.23 fold improvement in % α-guaiene relative toother products. FIG. 6 shows the fold improvement in titer of α-guaiene,based on expression of a variant having an F406L substitution inAcDGuaS3 (α-GS1) as compared to the parent enzyme.

Example 2: Production of Rotundone

Candidate α-guaiene oxidase enzymes where screened by co-expression inE. coli with FPPS and α-GS1. Production of rotundol and rotundone wereobserved with expression of an engineered Kaurene Oxidase (KOeng). See,US 2018/0135081, which is hereby incorporated by reference in itsentirety. Co-expression of Vitis vinifera dehydrogenase (VvDH) alongwith α-GS1, KOeng, and Camptotheca acuminata cytochrome P450 reductase(CaCPR) reduces the titer of rotundol (FIG. 7A) and increases rotundonetiter (FIG. 7B). Rotundone derived from oxidation of α-guaiene bycytochrome P450 was confirmed by GC/MS (FIGS. 8A and 8B). The KOeng canbe further engineered to improve specificity for the α-guaienesubstrate. An alignment with wild-type kaurene oxidase enzymes is shownin Example 10, which can assist this engineering.

FIG. 9 shows in vivo production of rotundol and rotundone using analternative CYP450 system, based on expression of Helianthus annuusgermacrene A monooxygenase (HaGAO). The E. coli strain includedexpression of α-GS1, engineered HaGAO for expression in E. coli (SEQ IDNO:52), and AaCPR (Artemisia annua cytochrome P450 reductase; SEQ ID NO:33). The fermentation was conducted in 96 well plates for 48 hours. Asshown in FIG. 9, the oxygenated product was substantially rotundone,with only minor amounts of the rotundol intermediate.

SEQUENCES Terpene Synthase Vitis vinifera VvGuaS SEQ ID NO: 1MSVPLSVSVTPILSQRIDPEVARHEATYHP NFWGDRFLHYNPDDDFCGTHACKEQQIQELKEEVRKSLEATAGNTSQLLKLIDSIQRLGL AYHFEREIEEALKAMYQTYTLVDDNDHLTTVSLLFRLLRQEGYHIPSDVFKKPMDEGGNF KESLVGDLPGMLALYEAAHLMVHGEDILDEALGFTTAHLQSMAIDSDNPLTKQVIRALKR PIRKGLPRVEARHYITIYQEDDSHNESLLKLAKLDYNMLQSLHRKELSEITKWWKGLDFA TKLPFARDRIVEGYFWILGVYFEPQYYLARRILMKVFGVLSIVDDIYDAYGTFEELKLFT EAIERWDASSIDQLPDYMKVCYQALLDVYEEMEEEMTKQGKLYRVHYAQAALKRQVQAYL LEAKWLKQEYIPTMEEYMSNALVTSACSMLTTTSFVGMGDMVTKEAFDWVFSDPKMIRAS NVICRLMDDIVSHEFEQKRGHVASAVECYMKQYGVSKEEAYDEFKKQVESAWKDNNEEVL QPTAVPVPLLTRVLNFSRMVDVLYKDEDEYTLVGPLMKDLVAGMLIDPVPM Pogostemon cablin PcPS (Q495P3) SEQ ID NO: 2MELYAQSVGVGAASRPLANFHPCVWGDKFI VYNPQSCQAGEREEAEELKVELKRELKEASDNYMRQLKMVDAIQRLGIDYLFVEDVDEAL KNLFEMFDAFCKNNHDMHATALSFRLLRQHGYRVSCEVFEKFKDGKDGFKVPNEDGAVAV LEFFEATHLRVHGEDVLDNAFDFTRNYLESVYATLNDPTAKQVHNALNEFSFRRGLPRVE ARKYISIYEQYASHHKGLLKLAKLDFNLVQALHRRELSEDSRWWKTLQVPTKLSFVRDRL VESYFWASGSYFEPNYSVARMILAKGLAVLSLMDDVYDAYGTFEELQMFTDAIERWDASC LDKLPDYMKIVYKALLDVFEEVDEELIKLGAPYRAYYGKEAMKYAARAYMEEAQWREQKH KPTTKEYMKLATKTCGYITLIILSCLGVEEGIVTKEAFDWVFSRPPFIEATLIIARLVND ITGHEFEKKREHVRTAVECYMEEHKVGKQEVVSEFYNQMESAWKDINEGFLRPVEFPIPL LYLILNSVRTLEVIYKEGDSYTHVGPAMQNIIKQLYLHPVPY Aquilaria crassna AcC2 (D0VMR6) SEQ ID NO: 3MSSAKLGSASEDVNRRDANYHPTVWGDFFL THSSNFLENNDSILEKHEELKQEVRNLLVVETSDLPSKIQLTDEIIRLGVGYHFETEIKA QLEKLHDHQLHLNFDLLTTSVWFRLLRGHGFSISSDVFKRFKNTKGEFETEDARTLWCLY EATHLRVDGEDILEEAIQFSRKKLEALLPELSFPLNECVRDALHIPYHRNVQRLAARQYI PQYDAEPTKIESLSLFAKIDFNMLQALHQRELREASRWWKEFDFFSKLPYARDRIAEGYY WMMGAHFEPKFSLSRKFLNRIIGITSLIDDTYDVYGTLEEVTLFTEAVERWDIEAVKDIP KYMQVIYTGMLGIFEDFKDNLINARGKDYCIDYAIEVFKEIVRSYQREAEYFHTGYVPSY DEYMENSIISGGYKMFIILMLIGRGEFELKETLDWASTIPEMVEASSLIARYIDDLQTYK AEEERGETVSAVRCYMREFGVSEEQACKKMREMIEIEWKRLNKTTLEADEISSSVVIPSL NFTRVLEVMYDKGDGYSDSQGVTKDRIAAL LRHAIEIAquilaria crassna AcC3 (D0VMR7) SEQ ID NO: 4MSSAKLGSASEDVSRRDANYHPTVWGDFFL THSSNFLENNDSILEKHEELKQEVRNLLVVETSDLPSKIQLTDEIIRLGVGYHFETEIKA QLEKLHDHQLHLNFDLLTTSVWFRLLRGHGFSIPSDVFKRFKNTKGEFETEDARTLWCLY EATHLRVDGEDILEEAIQFSRKRLEALLPKLSFPLSECVRDALHIPYHRNVQRLAARQYI PQYDAEQTKIESLSLFAKIDFNMLQALHQSELREASRWWKEFDFFSKLPYARDRIAEGYY WMMGAHFEPKFSLSRKFLNRIVGITSLIDDTYDVYGTLEEVTLFTEAVERWDIEAVKDIP KYMQVIYIGMLGIFEDFKDNLINARGKDYCIDYAIEVFKEIVRSYQREAEYFHTGYVPSY DEYMENSIISGGYKMFIILMLIGRGEFELKETLDWASTIPEMVKASSLIARYIDDLQTYK AEEERGETVSAVRCYMREFGVSEEQACKKMREMIEIEWKRLNKTTLEADEISSSVVIPSL NFTRVLEVMYDKGDGYSDSQGVTKDRIAAL LRHAIEIAquilaria crassna AcC4 (D0VMR8) SEQ ID NO: 5MSSAKLGSASEDVSRRDANYHPTVWGDFFL THSSDFLENNDSILEKHEELKQEVRNLLVVETSDLPSKIQLTDDIIRLGVGYHFETEIKA QLEKLHDHQLHLNFDLLTTSVWFRLLRGHGFSISSDVFKRFKNTKGEFETEDARTLWCLY EATHLRVDGEDILEEAIQFSRKKLEALLPELSFPLNECVRDALHIPYHRNVQRLAARQYI PQYDAEPTKIESLSLFAKIDFNMLQALHQRELREASRWWKEFDFFSKLPYARDRIAEGYY WMMGAHFEPKFSLSRKFLNRIIGITSLIDDTYDVYGTLEEVTLFTEAVERWDIEAVKDIP KYMQVIYIGMLGIFEDFKDNLINARGKDYCIDYAIEVFKEIVRSYQREAEYFHTGYVPSY DEYMENSIISGGYKMFIILMLIGRGEFELKETLDWASTIPEMVKASSLIARYIDDLQTYK AEEERGETVSAVRCYMREYDVSEEEACKKMREMIEIEWKRLNKTTLEADEVSSSVVIPSL NFTRVLEVMYDKGDGYSDSQGVTKDRIAAL LRHAIEIAquilaria crassna AcC1mut1-M42 SEQ ID NO: 6MSSAKLGSASEDVSRRDANYHPTVWGDFFL THSSNFLENNDSILEKHEELKQEVRNLLVVETSDLPSKIQLTDEIIRLGVGYHFETEIKA QLEKLHDHQLHLNFDLLTTSVWFRLLRGHGFSISSDVFKRFKNTKGEFETEDAWTLWCLY EATHLRVDGEDILEEAIQFSRKKLEALLPELSFPLNECVRDALHIPYHRNVQRLAARQYI PQYDAEPTKIESLSLFAKIDFNMLQALHQSELREASRWWKEFDFFSKLPYARDRIAEGYY WMMGAHFEPKFSLSRKFLNRIIGITSLIDDTYDVYGTLEEVTLFTEAVERWDIEAVKDIP KYMQVIYTGMLGIFEDFKDNLINARGKDYCIDYAIEVFKEIVRSYQREAEYFHTGYVPSY DEYMENSIISGGYKMFIILMLIGRGEFELKETLDWASTIPEMVKASSLIARYIDDLQTYK AEEERGETVSAVRCYMREFGVSEEQACKKMREMIEIEWKRLNKTTLEADEISSSVVIPSL NFTRVLEVMYDKGDGYSDSQGVTKDRIAAL LRHAIEIAquilaria crassna AcC1mut2-M50 SEQ ID NO: 7MSSAKLGSASEDVSRRDANYHPTVWGDFFL THSSNFLENNDSILEKHEELKQEVRNLLVVETSDLPSKIQLTDEIIRLGVGYHFETEIKA QLEKLHDHQLHLNFDLLTTSVWFRLLRGHGFSISSDVFKRFKNTKGEFETEDARTLWCLY EATHLRVDGEDILEEAIQFSRKKLEALLPELSFPLNECVRDALHIPYHRNVQRLAARQYI PQYDAEPTKIESLSLFAKIDFNMLQALHQSELREASRWWKEFDFFSKLPYARDRIAEGYY WMMGAHFEPKFSLSRKFLNRIIGITSLIDDTYDVYGTLEEVTLFTEAVERWDIEAVKDIP KYMQVIYTGMLGIFEDFKDNLINARGKDYCIDYAIEVFKEIVRSYQREAEYFHTGYVPSY DEYMENSIISGGYKMFIILMLIGRGEFELKETLDWASTIPEMVKASSLIARYIDDLQTYK AEEERGETVSAVRCYMREFGVSEEQACKKMREMIEIEWKRLNKTTLEADEISSSVVIPSL NFTRVLEVMYDKGDGYSDSQGVTKDRIAAL LRHAIEIAquilaria crassna AcDGuaS3 (F6LJD3) SEQ ID NO: 8MSSAKLGSASEDVSRRDANYHPTVWGDFFL THSSNFLENNDSILEKHEELKQEVRNLLVVETSDLPSKIQLTDEIIRLGVGYHFETEIKA QLEKLHDHQLHLNFDLLTTSVWFRLLRGHGFSISSDVFKRFKNTKGEFETEDARTLWCLY EATHLRVDGEDILEEAIQFSRKKLEALLPELSFPLNECVRDALHIPYHRNVQRLAARQYI PQYDAEPTKIESLSLFAKIDFNMLQALHQRELREASRWWKEFDFFSKLPYARDRIAEGYY WMMGAHFEPKFSLSRKFLNRIIGITSLIDDTYDVYGTLEEVTLFTEAVERWDIEAVKDIP KYMQVIYTGMLGIFEDFKDNLINARGKDYCIDYAIEVFKEIVRSYQREAEYFHTGYVPSY DEYMENSIISGGYKMFIILMLIGRGEFELKETLDWASTIPEMVKASSLIARYIDDLQTYK AEEERGETVSAVRCYMREFGVSEEQACKKMREMIEIEWKRLNKTTLEADEISSSVVIPSL NFTRVLEVMYDKGDGYSDSQGVTKDRIAAL LRHAIEIAquilaria crassna AcDGuaS4 (F6LJD4) SEQ ID NO: 9MSSAKLGSASEDVSRRDANYHPTVWGDFFL THSSNFLENNDNILEKHEELKQEVRNLLVVETSDLPSKIQLTDEIIRLGVGYHFEMEIKA QLEKLHDHQLHLNFDLLTTSVWFRLLRGHGFSISSDVFKRFKNTKGEFETEDARTLWCLY EATHLRVDGEDILEEAIQFSRKRLEALLPKLSFPLSECVRDALHIPYHRNVQRLAARQYI PQYDAEQTKIESLSLFAKIDFNMLQALHQSELREASRWWKEFDFFSKLPYARDRIAEGYY WMMGAHFEPKFSLSRKFLNRIIGITSLIDDTYDVYGTLEEVTLFTEAVERWDIEAVKDIP KYMQVIYIGMLGIFEDFKDNLINARGKDYCIDYAIEVFKEIVRSYQREAEYFHTGYVPSY DEYMENSIISGGYKMFIILMLIGRGEFELKETLDWASTIPEMVKASSLIARYIDDLQTYK AEEERGETVSAVRCYMREYDVSEEEACKKMREMIEIEWKRLNKTTLEADEVSSSVVIPSL NFTRVLEVMYDKGDGYSDSQGVTKDRIAAL LRHAIEIAquilaria crassna AcDGuaS2 (F6LJD2) SEQ ID NO: 10MSSAKLGSASEDVSRRDANYHPTVWGDFFL THSSNFLENNDSILEKHEELKQEVRNLLVVETIDLPSKIQLTDEIIRLGVGYHFEMEIKA QLEKLHDHQLHLNFDLLTTSVWFRLLRGHGFSISSDVFKRFKNTKGEFETEDARTLWCLY EATHLRVDGEDILEEAIQFSRKKLEALLPELSFPLNECVRDALHIPYHLNVQRLAARQYI PQYDAEPTKIESLSLFAKIDFNMLQALHQSELREASRWWKEFDFFSKLPYARDRIAEGYY WMMGAHFEPKFSLSRKFLNRIIGITSLIDDTYDVYGTLEEVTLFTEAVERWDIEAVKDIP KYMQVIYTGMLGIFEDFKDNLINARGKDYCIDYAIEVFKEIVRSYQREAEYFHTGYVPSY DEYMENSIISGGYKMFIILMLIGRGEFELKETLDWASTIPEMVKASSLIARYIDDLQTYK AEEERGETVSAVRCYMREYGVSEEEACKKMREMIEIEWKRLNKTTLEADEISSSVVIPSL NFTRVLEVMYDKGDGYSDSQGVTKDRIAAL LRHAIEIAquilaria crassna AcDGuaS5 (F6LJD5) SEQ ID NO: 11MSSAKLGSASEDVSRRDANYHPTVWGDFFL THSSNFLENNDNILEKHEELKQEVRNLLVVETSDLPSKIQLTDEIIRLGVGYHFEMEIKA QLEKLHDHQLHLNFDLLTTSVWFRLLRGHGFSISSDVFKRFKNTKGEFETEDARTLWCLY EATHLRVDGEDILEEAIQFSRKRLEALLPKLSFPLSECVRDALHIPYHRNVQRLAARQYI PQYDAEQTKIESLSLFAKIDFNMLQALHQSELREASRWWKEFDFFSKLPYARDRIAEGYY WMMGAHFEPKFSLSRKFLNRIIGITSLIDDTYDVYGTLEEVTLFTEAVERWDIEAVKDIP KYMQVIYIGMLGIFEDFKDNLINARGKDYCIDYAIEVFKEIVRSYQREAEYFHTGYVPSY DEYMENSIISGGYKMFIILMLIGRGEFELKQTLDWASTIPEMVKASSLIARYIDDLQTYK AEEERGETVSAVRCYMREYDVSEEEACKKMREMIEIEWKRLNKTTLEADEVSSSVVIPSL NFTRVLEVMYDKGDGYSDSQGVTKDRIAAL LRHAIEIAquilaria spp. AmiDGuaS1 (A0A0U3ACM2) SEQ ID NO: 12MSSAKLGSASEDVSRRDANYHPTVWGDFFL THSSNFLENNDNILEKHEELKQEVRNLLVVETSDLPSKIQLTDKIIRLGVGYHFEMEIKA QLEKLHDHQLHLNFDLLTTSVWFRLLRGHGFSISSDVFKRFKNTKGEFETEDARTLWCLY EATHLRVDGEDILEEAIQFSRKKLEALLPELSFPLNECVRDALHIPYHRNVQRLAARQYI PQYDAELTKIESLSLFAKIDFNMLQALHQSELREASRWWKEFDFFSKLPYARDRIAEGYY WMMGAHFEPKFSLSRKFLNRIIGITSLIDDTYDVYGTLEEVTLFTKAVERWDIEAVQDIP KYMQVIYTGMLGIFEDFKDNLINARGKDYCIDYAIEVFKEIVRSYQREAEYFHTGYVPSY DEYMENSIISGGYKMFIILMLIGRGEFELKETLDWASTIPEMVKASSLIARYIDDLQTYK AEEKRGETVSAVRCYMREYGVSEEEACKKMREMIEIEWKKLNKTTLEANEISSSVVIPSL NFTRVLEVMYDKGDGYSDSQGVTKDRIAAL LRHAIEIAquilaria spp. AmiDGuaS2 (A0A0U3A773) SEQ ID NO: 13MSSAKLGSASEDVSRRDANYHPTVWGDFFL THSSNFLENNDNILEKHEELKQEVTNLLVVETSDLPSKIQLTDEIIRLGVGYHFEMEIKA QLEKLHDHQLHLNFDLLTTSVWFRLLRGHGFSISSDVFKRFKNTKGEFETEDARTLWCLY EATHLRVDGEDILEEAIQFSRKKLEALLPELSFPLNECVRDALHIPYHRNVQRLAARQYI PQYDAELTKIESLSLFAKIDFNMLQALHQSELREASRWWKEFDFFSKLPYARDRIAEGYY WMMGAHFEPKFSLSRKFLNRIIGITSLIDDTYDVYGTLEEVTLFTEAVERWDIEAVKDIP KYMQVIYTGMLGIFEDFKDNLINARGKDYCIDYAIEVFKEIVRSYQREAEYFHTGYVPSY DEYMENSIISGGYKMFIILMLIGRAEFELKETLDWASTIPEMVKASSLIARYIDDLQTYK AEEERGETVSAVRCYMREYGVSEEEACKKMREMIEIEWKRLNKTTLEADEISSSVVIPSL NFTRVLEVMYDKGDGYSDSQGVTKGRIAAL LRHAIEIAquilaria spp. AmiDGuaS3 (A0A023J8Z5 SEQ ID NO: 14MSSAKLGSASEDVSRRDANYHPTVWGDFFL THSSNFLENNHSILEKHEELKQEVRNLLVVETSDLPSKIQLTDKIIRLGVGYHFEMEIKA QLEKLHDHQLHLNFDLLTTSVWFRLLRGHGFSISSDVFKRFKNTKGEFETEDARTLWCLY EATHLRVDGEDILEEAIQFSRKKLEALLPELSFPLNECVRDALHIPYHRNVQRLAARQYI PQYDAELTKIESLSLFAKIDFNMLQALHQSELREASRWWKEFDFFSKLPYARDRIAEGYY WMMGAHFEPKFSLSRKFLNRIIGITSLIDDTYDVYGTLEEVTLFTEAVERWDIEAVKDIP KYMQVIYTGMLGIFEDFKDNLINARGKDYCIDYAIEVFKEIVRSYQREAEYFHTGYVPSY DEYMENSIISGGYKMFIILMLIGRAEFELKETLDWASTIPEMVKASSLIARYIDDLQTYK AEEERGETVSAVRCYMREYGVSEEEACKKMREMIEIEWKRLNKTTLEADEISSSVVIPSL NFTRVLEVMYDKGDGYSDSQGVTKDRIAAL LRHAIEIAquilaria spp. AmaDGuaS1 (A0A1B0U478) SEQ ID NO: 15MSSAKLGSAPEDVSRRDANYHPTVWGDFFL THSSNFLENNHSILEKHEELKQEVRNLLVVETSDLPSKIQLTDKIIRLGVGYHFEMEIKA QLEKLQDHQLHLNFDLLTTSVWFRLLRGHGFSISSDVFKRFKNTKGEFETEDARTLWCLY EATHLRVDGEDILEEAIQFSRKKLEALLPELSFPLNECVRDALHIPYHRNVQRLAARQYI PQYDAELTKIESLSLFAKIDFNMLQALHQSELREASRWWKEFDFFSKLPYARDRIAEGYY WMMGAHFEPKFSLSRKFLNRIIGITSLIDDTYDVYGTLEEVTLFTEAVERWDIEAVKDIP KYMQVIYTGMLGIFEDFKDNLINARGKDYCIDYAIEVFKEIVRSYQREAEYFHTGYVPSY DEYMENSIISGGYKMFIILMLIGRAEFELKETLDWASTIPEMVKASSLIARYIDDLQTYK AEEERGETVSAVRCYMREYGVSEEEACKKMREMIEIEWKRLNKTTLEADEISSSVVIPSL NFTRVLEVMYDKGDGYSDSQGVTKDRIATL LRHAIEIAquilaria spp. AmaDGuaS2 (A0A0U2YQ77) SEQ ID NO: 16MSSAKLGSASEDVSRRDADYHPTVWGDFFL THSSNFLENNHSILEKHEELKQEVRNLLVVETSDLPSKIQLTDKIIRLGVGYHFEMEIKA QLEKLHDHQLHLNFDLLTTSVWFRLLRGHGFSISSDVFKRFKNTKGEFETEDARTSWCLY EATHLRVDGEDILEEAIQFSRKKLEALLPELSFPLNECVRDALHIPYHRNVQRLAARQYI SQYDAELTKIESLSLFAKIDFNMLQALHQSELREASRWWKEFDFFSKLPYARDRIAEGYY WMMGAHFEPKFSLSRKFLNRIIGITSLIDDTYDVYGTLEEVTLFTEAVERWDIEAVKDIP KYMQVIYTGMLGIFEDFKDNLINARGKDYCIDYAIEVFKEIVRSYQREAEYFHTGYVPSY DEYMENSIISGGYKMFIILMLIGRAEFELKETLDWASTIPEMVKASSLIARYIDDLQTYK AEEERGETVSAVRCYMREYGVSEEEACKKMREMIEIEWKRLNKTTLEADEISSSVVIPSL NFTRVLEVMYDKGDGYSDSQGVTKDRIAAquilaria spp. AsDGuaS1 (K9MQ67) SEQ ID NO: 17MSSAKLGSTSEDVSRRDANYHPTVWGDFFL THSSNFLENNDSILEKHEELKQEVRNLLVVETSDLPSKIQLTDEIIRLGVGYHFETEIKA QLEKLHDHQLHLNFDLLTTSVWFRLLRGHGFSISSDVFKRFKNTKGEFKTEDARTLWCLY EATHLRVDGEDVLEEAIQFSRKKLEALLPELSFPLSECVRDALHIPYHRNVQRLAARQYI PQYDAEPTKIESLSLFAKIDFNMLQALHQSELREASRWWKEFDFFSKLPYARDSIAEGYY WMMGAHFEPKFSLSRKFLNRIIGITSLIDDTYDVYGTLEEVTLFTEAVERWDIEAVKDIP KYMQVIYTGMLGIFEDFKDNLINARGKDYCIDYAIEVFKEIVRSYQREAEYFHTGYVPSY DEYMENSIISGGYKMFIILMLIGRGEFELKETLDWASTIPEMVKASSLIARYIDDLQTYK AEEKRGETVSAVRCYMREYGVSEEEACKKMKEMIEIEWKRLNKTTLEADEISSSVVIPSL NFTRVLEVMYDKGDGYSDSQGVTKDRIAAL LRHAIEIAquilaria spp. AsDGuaS2 (K9MNV6) SEQ ID NO: 18MSSAKLGSASEDVSRRDANYHPTVWGDFFL THSSNFLENNDSILEKHEELKQEVRNLLVVETSDLPSKIQLTDEIIRLGVGYHFETEIKA QLEKLHDHQLHLNFDLLTTSVWFRLLRGHGFSISSDVFKRFKNTKGEFKTEDARTLWCLY EATHLRVDGEDVLEEAIQFSRKKLEALLPELSFPLSECVRDALHIPYHRNVQRLAARQYI PQYDAEPTKIESLSLFAKIDFNMLQALHQSELREASRWWKEFDFFSKLPYARDRIAEGYY WMMGAHFEPKFSLSRKFLNRIIGITSLIDDTYDVYGTLEEVTLFTEAVERWDIEAVKDIP KYMQVIYTGMLGIFEDFKDNLINARGKDYCIDYAIEVFKEIVRSYQREAEYFHTGYVPSY DEYMENSIISGGYKMFIILMLIGRGEFELKETLDWASTIPEMVKASSLIARYIDDLQTYK AEEKRGETVSAVRCYMREYGVSEEEACKKMKEMIEIEWKRLNKTTLEADEISSSVVIPSL NFTRVLEVMYDKGDGYSDSQGVTKDRIAAL LRHAIEIAquilaria spp. AsDGuaS3 (K9MPP8) SEQ ID NO: 19MSSAKLGSASEDVSRRDANYHPTVWGDFFL THSSNFLENNDSILEKHEELKQEVRNLLVVETSDLPSKIQLTDEIIRLGVGYHFETEIKA QLEKLHDHQLHLNFDLLTTSVWFRLLRGHGFSISSDVFKRFKNTKGEFKTEDARTLWCLY EATHLRVDGEDVLEEAIQFSRKKLEALLPELSFPLSECVRDALHIPYHRNVQRLAARQYI PQYDAEPTKIESLSLFAKIDFNMLQALHQSELREASRWWKEFDFFSKLPYARDRIAEGYY WMMGAHFEPKFSLSRKFLNRIIGITSLIDDTYDVYGTLEEVTLFTEAVERWDIEAVKDIP KYMQVIYTGMLGIFEDFKDNLINARGKDYCIDYAIEVFKEIVRSYQREAEYFHTGYVPSY DEYMENSIISGGYKMFIILMLIGRGEFELKETLDWASTIPEMVKASSLIARYIDDLQTYK AEEERGETVSAVRCYMREYGVSEEEACKKMREMIEIEWKRLNKTTLEADEISSSVVIPSL NFTRVLEVMYDKGDGYSDSQGVTKDRIAAL LRHAIEIAquilaria spp. AsDGuaS4 (M9SVT6) SEQ ID NO: 20MSSAKLGSASEDVSRRDANYHPTVWGDFFL THSSNFLENNDNILEKHEELKQEVRNLLVVETSDLPSKIQLTDEIIRLGVGYHFEMEIKA QLEKLHDHQLHLNFDLLTTSVWFRLLRGHGFSISSDVFKRFKNTKGEFETEDARTLWCLY EATHLRVDGEDILEEAIQFSRKRLEALLPKLSFPLSECVRDALHIPYHRNVQRLAARQYI PQYDAEQTKIESLSLFAKIDFNMLQALRQSELREASRWWKEFDFFSKLPYARDRIAEGYY WMMGAHFEPKFSLSRKFLNRIIGITSLIDDTYDVYGTLEEVTLFTEAVERWDIEAVKDIP KYMQVIYTGMLGIFEDFKDNLINARGKDYCIDYAIEVFKEIVRSYQREAEYFHTGYVPSY DEYMENSIISGGYKMFIILMLIGRGEFELKETLDWASTIPEMVKASSLIARYIDDLQTYK AEEERGETVSAVRCYMREFGVSEEQACKKMREMIEIEWKRLNKTTLEADEISSSVVIPSL NFTRVLEVMYDKGDGYSDSQGVTKDRIAAL LRHAIEIV. vinifera germacrene D synthase (VvGDS) SEQ ID NO: 21MSVPLSVSVTPILSQRIDPEVARHEATYHP NFWGDRFLHYNPDDDFCGTHACKEQQIQELKEEVRKSLEATAGNTSQLLKLIDSIQRLGL AYHFEREIEEALKAMYQTYTLVDDNDHLTTVSLLFRLLRQEGYHIPSDVFKKFMDEGGNF KESLVGDLPGMLALYEAAHLMVHGEDILDEALGFTTAHLQSMAIDSDNPLTKQVIRALKR PIRKGLPRVEARHYITIYQEDDSHNESLLKLAKLDYNMLQSLHRKELSEITKWWKGLDFA TKLPFARDRIVEGYFWILGVYFEPQYYLARRILMKVFGVLSIVDDIYDAYGTFEELKLFT EAIERWDASSIDQLPDYMKVCYQALLDVYEEMEEEMTKQGKLYRVHYAQAALKRQVQAYL LEAKWLKQEYIPRMDEYMSNALVSSACSMLTTTSFVGMGDIVTKEAFDWVFSDPKMIRAS NVICRLMDDIVSHEFEQKRGHVASAVECYMKQYGVSKEEAYDEFKKQVESAWKDNNEEFL QPTAVPVPLLTRVLNFSRMMDVLYKDEDEYTLVGPLMKDLVAGMLIDPVPM α-Guaiene Oxidase V. viniferaVvSTO2 (F6I534; Engineered CYP71BE5 α-guaiene 2-oxidase) SEQ ID NO: 22MELQFSFFPILCTFLLFIYLLKRLGKPSRT NHPAPKLPPGPWKLPIIGNMHQLVGSLPHRSLRSLAKKHGPLMHLQLGEVSAIVVSSREM AKEVMKTHDIIFSQRPCILAASIVSYDCTDTAFAPYGGYWRQIRKISVLELLSAKRVQSF RSVREEEVLNLVRSVSLQEGVLINLIKSIFSLTFSIISRTAFGKKCKDQEAFSVTLDKFA DSAGGFTIADVFPSIKLLHVVSGMRRKLEKVHKKLDRILGNIINEHKARSAAKETCEAEV DDDLVDVLLKVQKQGDLEFPLTMDNIKAVLLDLFVAGTETSSTAVEWAMAEMLKNPRVMA KAQAEVRDIFSRKGNADETVVRELKFLKLVIKETLRLHPPVPLLIPRESRERCAINGYEI PVKTRVIINAWAIARDPKYWTDAESFNPERFLDSSIDYQGTNFEYIPFGAGRRMCPGILF GMANVELALAQLLYHFDWKLPNGARHEELDMTEGFRTSTKRKQDLYLIPITYRPLPVE B. subtilis BsAGOX1 (O31440; cypC CYP152A1)SEQ ID NO: 23 MNEQIPHDKSLDNSLTLLKEGYLFIKNRTERYNSDLFQARLLGKNFICMTGAEAAKVFYD TDRFQRQNALPKRVQKSLFGVNAIQGMDGSAHIHRKMLFLSLMTPPHQKRLAELMTEEWK AAVTRWEKADEVVLFEEAKEILCRVACYWAGVPLKETEVKERADDFIDMVDAFGAVGPRH WKGRRARPRAEEWIEVMIEDARAGLLKTTSGTALHEMAFHTQEDGSQLDSRMAAIELINV LRPIVAISYFLVFSALALHEHPKYKEWLRSGNSREREMFVQEVRRYYPFGPFLGALVKKD FVWNNCEFKKGTSVLLDLYGTNHDPRLWDHPDEFRPERFAEREENLFDMIPQGGGHAEKG HRCPGEGITIEVMKASLDFLVHQIEYDVPEQSLHYSLARMPSLPESGFVMSGIRRKS B. subtilis BsAGOX1 (A5HNX5; pksS CYP107K1)SEQ ID NO: 24 MQMEKLMFHPHGKEFHHNPFSVLGRFREEEPIHRFELKRFGATYPAWLITRYDDCMAFLK DNRITRDVKNVMNQEQIKMLNVSEDIDFVSDHMLAKDTPDHTRLRSLVHQAFTPRTIENL RGSIEQIAEQLLDEMEKENKADIMKSFASPLPFIVISELMGIPKEDRSQFQIWTNAMVDT SEGNRELTNQALREFKDYIAKLIHDRRIKPKDDLISKLVHAEENGSKLSEKELYSMLFLL VVAGLETTVNLLGSGTLALLQHKKECEKLKQQPEMIATAVEELLRYTSPVVMMANRWAIE DFTYKGHSIKRGDMIFIGIGSANRDPNFFENPEILNINRSPNRHISFGFGIHFCLGAPLA RLEGHIAFKALLKRFPDIELAVAPDDIQWRKNVFLRGLESLPVSLSK B. cereus BcAGOX1 (Q737I9; BCE_2659 CYP106)SEQ ID NO: 25 MASPENVILVHEISKLKTKEELWNPYEWYQFMRDNHPVHYDEEQDVWNVFLYEDVNRVLS DYRLFSSRRERRQFSIPPLETRININSTDPPEHRNVRSIVSKAFTPRSLEQWKPRIQAIA NELVQHIGKYSEVNIVEEFAAPLPVTVISDLLGVPTTDRKKIKAWSDILFMPYSKEKFND LDVEKGIALNEFKAYLLPIVQEKRYHLTDDIISDLIRAEYEGERLTDEEIVTFSLGLLAA GNETTTNLIINSFYCFLVDSPGTYKELREEPTLISKAIEEVLRYRFPITLARRITEDTNI FGPLMKKDQMVVAWVSAANLDEKKFSQASKFNIHRIGNEKHLTFGKGPHFCLGAPLARLE AEIALTTFINAFEKIALSPSFNLEQCILENEQTLKFLPICLKTQ B. cereus BcAGOX2 (Q737F3; cypA BCE_2696 CYP107)SEQ ID NO: 26 MKKLTFNDLNSPETMRNPIMFYKNLMEQKERFFHIDDFYGMGGAWVVFHYDDVVAILKDS RFIKDLRKFTPPHYKQNPIEENTAVSKLFEWLMNMPNMLTVDPPDHTRLRRLVSKSFTPR MIEDLRPRIQQIADELLDVVQEQRKMEIIADFAYPLPIIVISEMLGIPATDRNQFRAWTQ ELMKASVDPGQGTTVTATLEKFINYIEILFNEKHLNPSDDLISALVQAKEQEDKLSKNEL LSTIWLLIIAGHETTVNLISNGVLALLQHPEQMNLLRQDPSLLASAVDELLRYAGPIMFS SRFASEDVTIHGNRIRKGELVLLSLTAANIDPNIFPYPEELNISREENNHLAFGAGIHQC LGAPLARLEGQIALDTLLKRLPNLRLAIEADQLIYNHSKIRSLASLPVIF Nicotiana tomentosiformis NtKO SEQ ID NO: 45MDAILNLQTVPLGTALTIGGPAVALGGISL WFLKEYVNDQKRKSSNFLPPLPEVPGLPVIGNLLQLTEKKPHKTFTNWAETYGPIYSIKT GANTIVVLNTNELAKEAMVTRYSAISTRKLTNALKILTCDKSIVAISDYDEFHKTVKRHV LTSVLGPNAQKRHRIHRDTLIENVSKQLHDLVRKYPNEAVNLRKIFQSELFGLALKQALG KDIESIYVEGLDATLPREDVLKTLVLDIMEGAIDVDWRDFFPYLKWVPNKSFENRIQRKH LRREAVMKALIMEQRKRINSGEKLNSYIDYLSSEANTLTEKQILMLLWEAIIETSDTTVV STEWAMYELAKDPKRQEQLFLEIQNVCGSNKITEEKLCQLPYLCAVFHETLRKHSPVPIV PLRYVHEDTQLGGYHIPKGAEIAINIYGCNRDKKVWESPEEWKPERFLDGKYDPVELQKT MAFGGGKRVCAGALQAMTITCTTIARLIQEFEWSLKDGEEENVATMGLTTHKLHPMQAHI KPRK Lactuca sativa LsKO SEQ ID NO: 46MDGVIDMQTIPLRTAIAIGGTAVALVVALY FWFLRSYASPSHHSNHLPPVPEVPGVPVLGNLLQLKEKKPYMTFTKWAEMYGPIYSIRTG ATSMVVVSSNEIAKEVVVTRFPSISTRKLSYALKVLTEDKSMVAMSDYHDYHKTVKRHIL TAVLGPNAQKKFRAHRDTMMENVSNELHAFFEKNPNQEVNLRKIFQSQLFGLAMKQALGK DVESIYVKDLETTMKREEIFEVLVVDPMMGAIEVDWRDFFPYLKWVPNKSFENIIHRMYT RREAVMKALIQEHKKRIASGENLNSYIDYLLSEAQTLTDKQLLMSLWEPIIESSDTTMVT TEWAMYELAKNPNMQDRLYEEIQSVCGSEKITEENLSQLPYLYAVFQETLRKHCPVPIMP LRYVHENTVLGGYHVPAGTEVAINIYGCNMDKKVWENPEEWNPERFLSEKESMDLYKTMA FGGGKRVCAGSLQAMVISCIGIGRLVQDFEWKLKDDAEEDVNTLGLTTQKLHPLLALINP RK Cynara cardunculus var. scolymus CcKOSEQ ID NO: 47 MDMQSIPAIAIGSTAVAIALGLFFWFFRRHVPDHIDHPNHLPSVPEVPGIPVLGNLLQLK EKKPYMTFTKWAETYGPIYSIRTGAISMVVVSSNAIAKEALVTRFPSISTRKLSKALEVL TADKTMVAMSDYNDYHKTVKRHILTAVLGPNAQKKHRVHRDIMMQNLSNQLHTFVQNSPQ EEVNLRKVFQSELFGLAMRQTMGKDVESIYVEDLGTTMNRDEIFQVLVVDPLMGAIEVDW RDFFPYLKWIPNRNFENTIQQMYIRREAVMKALIQEHRKRIASGENLNSYIDYLLSEAQT LSEKQLXMSLWEPIIESSDTTMVTTEWAMYELAKNPKIQDRLYREIQGVCGSDKIXEENL GQLPYLSAIFNETLRRHGPVPIIPLRYVHEDTELGGYHIPAGTQIAVNIYGCNMEKAVWE NPEEWNPERFFEVEGDQKTMAFGGGKRVCAGSLQAMLIACIGIGRMVQEFEWKLKDEAAQ EDVNTLGLTTQKLRPLHAIIYPRKENDAKV WKCArtemisia annua AaKO SEQ ID NO: 49 MDALTDMLQIPPATPITVAITTVTIAVAIFLYIKSHASNHSRRSTHLPPVPEVPGVPVLG NLLQLKEKKPYLTFTRWAQTYGAIYSIRTGATSMVVVSSSEIAKEAMVTRFSSISTRNLS KALTILTADKTMVAMSDYNDYHRTVKRHILTAMLGPNAQRKQRVHRDPMIENISKQLHAF VENSPKEEVDLRKIFQSELFGLAMKQAVGKDVESLNVEDLGVTMKRDEIFQVLVVDPMMG AIEVDWRDFFPYLKWVPNKKFENTIQQMYIRRKAVMKALIKEHKKRIASGENLNSYIDYL LSEAQTFTDEQLIMSLWEPIIESSDTTMVTTEWAMYELAKNPKMQDRLYRDIQSVCGSDK ITEENLSQLPYLSAIFHETLRRHSPVPIIPLRHVHEDTVLGGYHVPAGTELAVNIYGCNM EKNVWENPEEYNPDRFMKENETIDMQRTMAFGGGKRVCAGSLQAMLISCIGIGRMVQEFE WRFKDKAEEDINTLGLTTQRLNPLRAIIKP RNHelianthus annuus HaKO SEQ ID NO: 50 MDALTGMLPIPPATALAIGGTAIALAVAISFWFLRSYTSGESNRLPRVPEVPGVPVLGNL LQLKEKKPYMTFTRWAETYGPIYSIRTGATSMVVVSSNEIAKEAFVTRFESISTRNLSKA LKILTDDKTMVAMSDYNDYHKTVKRHILTAMLGPNAQKKHRIQRDIMMENLSNRLHAFVK TSTEQEEVDLREIFQSELFGLAMRQTMGKDVESIYVEDLKITMKRDEIFQVLVVDPMMGA IDVDWRDFFPYLKWVPNKKFENTIQQMYIRREAVMKALIKQHKERIASGEKLNSYIDYLL SEAQSLTDRQLLMSVWEPIIESSDTTMVTTEWAIYELAKNPHIQDRLYRDIQSVCGSDII KEEHLSQLPFITAIFHETLRRHSPVPIIPLRYVHEDTVLGGYHVPAGTELAINIYGCNME KSVWENPEEWNPERFMKENETIDFQKTMAFGGGKRVCAGSLQAMLISCVGIGRMVQEFKW ELKNKAQEEVNTIGLTTQMLRPLRAIIKPR NEngineered Kaurene Oxidase (KOeng) SEQ ID NO: 51MAWEYALIGLVVGIIIGAVAMRWYLKSYTS ARRSQSNHLPRVPEVPGVPLLGNLLQLKEKKPYMTFTKWAATYGPIYSIKTGATSVVVVS SNEIAKEALVTRFQSISTRNLSKALKVLTADKQMVAMSDYDDYHKTVKRHILTAVLGPNA QKKHRIHRDIMMDNISTQLHEFVKNNPEQEEVDLRKIFQSELFGLAMRQALGKDVESLYV EDLKITMNRDEILQVLVVDPMMGAIDVDWRDFFPYLKWVPNKKFENTIQQMYIRREAVMK SLIKEQKKRIASGEKLNSYIDYLLSEAQTLTDQQLLMSLWEPIIESSDTTMVTTEWAMYE LAKNPKLQDRLYRDIKSVCGSEKITEEHLSQLPYITAIFHETLRKHSPVPILPLRHVHED TVLGGYHVPAGTELAVNIYGCNMDKNVWENPEEWNPERFMKENETIDFQKTMAFGGGKRV CAGSLQALLIASIGIGRMVQEFEWKLKDMTQEEVNTIGLTNQMLRPLRAIIKPRI Helianthus annuus germacrene Amonooxygenase; Engineered HaGAO SEQ ID NO: 52MAKPPLFFIVIIGLIVVAASFLYKLLTRPT SSKNRLPEPWRLPIIGHMHHLIGTMPHRGVMDLARKYGSLMHLQLGEVSAIVVSSPKWAK EILTTYDIPFANRPETLTGEIIAYHNTDIVLAPYGEYWRQLRKLCTLELLSVKKVKSFQS LREEECWNLVQEIKASGSGTPFNLSEGIFKVIATVLSRAAFGKGIKDQKQFTEIVKEILR ETGGFDVADIFPSKKFLHHLSGKRGRLTSIHNKLDSLINNLVAEHTVSKSSKVNETLLDV LLRLKNSEEFPLTADNVKAIILDMFGAGTDTSSATVEWAISELIRCPRAMEKVQAELRQA LNGKERIKEEEIQDLPYLNLVIRETLRLHPPLPLVMPRECRQAMNLAGYDVANKTKLIVN VFAINRDPEYWKDAESFNPERFENSNTTIMGADYEYLPFGAGRRMCPGSALGLANVQLPL ANILYYFKWKLPNGASHDQLDMTESFGATVQRKTELMLVPSF Alpha-humulene 10-hydroxylase; Engineered CYP71BA1SEQ ID NO: 54 MAQDLRLILIIVGAIAIIALLVHGFLLIKRSSRSSVHKQQVLLASLPPSPPRLPLIGNIH QLVGGNPHRILLQLARTHGPLICLRLGQVDQVVASSVEAVEEIIKRHDLKFADRPRDLTF SRIFFYDGNAVVMTPYGGEWKQMRKIYAMELLNSRRVKSFAAIREDVARKLTGEIAHKAF AQTPVINLSEMVMSMINAIVIRVAFGDKCKQQAYFLHLVKEAMSYVSSFSVADMYPSLKF LDTLTGLKSKLEGVHGKLDKVFDEIIAQRQAALAAEQAEEDLIIDVLLKLKDEGNQEFPI TYTSVKAIVMEIFLAGTETSSSVIDWVMSELIKNPKAMEKVQKEMREAMQGKTKLEESDI PKFSYLNLVIKETLRLHPPGPLLFPRECRETCEVMGYRVPAGARLLINAFALSRDEKYWG SDAESFKPERFEGISVDFKGSNFEFMPFGAGRRICPGMTFGISSVEVALAHLLFHFDWQL PQGMKIEDLDMMEVSGMSATRRSPLLVLAK LIIPLPEnt-isokaurene C2-hydroxylase; Engineered CYP71Z18 SEQ ID NO: 55MAQDLRLILIIVGAIAIIALLVHGFLKSAV TKPKLNLPPGPWTLPLIGSIHHIVSNPLPYRAMRELAHKHGPLMMLWLGEVPTLVVSSPE AAQAITKTHDVSFADRHINSTVDILTFNGMDMVFGSYGEQWRQLRKLSVLELLSAARVQS FQRIREEEVARFMRSLAASASAGATVDLSKMISSFINDTFVRESIGSRCKYQDEYLAALD TAIRVAAELSVGNIFPSSRVLQSLSTARRKAIASRDEMARILGQIIRETKESMDQGDKTS NESMISVLLRLQKDAGLPIELTDNVVMALMFDLFGAGSDTSSTTLTWCMTELVRYPATMA KAQAEVREAFKGKTTITEDDLSTANLRYLKLVVKEALRLHCPVPLLLPRKCREACQVMGY DIPKGTCVFVNVWAICRDPRYWEDAEEFKPERFENSNLDYKGTYYEYLPFGSGRRMCPGA NLGVANLELALASLLYHFDWKLPSGQEPKDVDVWEAAGLVAKKNIGLVLHPVSHIAPVNA Premnaspirodiene oxygenase;Engineered CYP71D55_V482I/A484I SEQ ID NO: 56MAQDLRLILIIVGAIAIIALLVHGFFLLRK WKNSNSQSKKLPPGPWKLPLLGSMLHMVGGLPHHVLRDLAKKYGPLMHLQLGEVSAVVVT SPDMAKEVLKTHDIAFASRPKLLAPEIVCYNRSDIAFCPYGDYWRQMRKICVLEVLSAKN VRSFSSIRRDEVLRLVNFVRSSTSEPVNFTERLFLFTSSMTCRSAFGKVFKEQETFIQLI KEVIGLAGGFDVADIFPSLKFLHVLTGMEGKIMKAHHKVDAIVEDVINEHKKNLAMGKTN GALGGEDLIDVLLRLMNDGGLQFPITNDNIKAIIFDMFAAGTETSSSTLVWAMVQMMRNP TILAKAQAEVREAFKGKETFDENDVEELKYLKLVIKETLRLHPPVPLLVPRECREETEIN GYTIPVKTKVMVNVWALGRDPKYWDDADNFKPERFEQCSVDFIGNNFEYLPFGGGRRICP GISFGLANVYLPLAQLLYHFDWKLPTGMEPKDLDLTELVGITIARKSDLMLVATPYQPS RE Cytochrome P450 ReductaseStevia rebaudiana SrCPR SEQ ID NO: 27 MAQSDSVKVSPFDLVSAAMNGKAMEKLNASESEDPTTLPALKMLVENRELLTLFTTSFAV LIGCLVFLMWRRSSSKKLVQDPVPQVIVVKKKEKESEVDDGKKKVSIFYGTQTGTAEGFA KALVEEAKVRYEKTSFKVIDLDDYAADDDEYEEKLKKESLAFFFLATYGDGEPTDNAANF YKWFTEGDDKGEWLKKLQYGVFGLGNRQYEHFNKIAIVVDDKLTEMGAKRLVPVGLGDDD QCIEDDFTAWKELVWPELDQLLRDEDDTSVTTPYTAAVLEYRVVYHDKPADSYAEDQTHT NGHVVHDAQHPSRSNVAFKKELHTSQSDRSCTHLEFDISHTGLSYETGDHVGVYSENLSE VVDEALKLLGLSPDTYFSVHADKEDGTPIGGASLPPPFPPCTLRDALTRYADVLSSPKKV ALLALAAHASDPSEADRLKFLASPAGKDEYAQWIVANQRSLLEVMQSFPSAKPPLGVFFA AVAPRLQPRYYSISSSPKMSPNRIHVTCALVYETTPAGRIHRGLCSTWMKNAVPLTESPD CSQASIFVRTSNFRLPVDPKVPVIMIGPGTGLAPFRGFLQERLALKESGTELGSSIFFFG CRNRKVDFIYEDELNNFVETGALSELIVAFSREGTAKEYVQHKMSQKASDIWKLLSEGAY LYVCGDAKGMAKDVHRTLHTIVQEQGSLDSSKAELYVKNLQMSGRYLRDVW Arabidopsis thaliana AtCPR1 SEQ ID NO: 28MATSALYASDLFKQLKSIMGTDSLSDDVVL VIATTSLALVAGFVVLLWKKTTADRSGELKPLMIPKSLMAKDEDDDLDLGSGKTRVSIFF GTQTGTAEGFAKALSEEIKARYEKAAVKVIDLDDYAADDDQYEEKLKKETLAFFCVATYG DGEPTDNAARFYKWFTEENERDIKLQQLAYGVFALGNRQYEHFNKIGIVLDEELCKKGAK RLIEVGLGDDDQSIEDDFNAWKESLWSELDKLLKDEDDKSVATPYTAVIPEYRVVTHDPR FTTQKSMESNVANGNTTIDIHHPCRVDVAVQKELHTHESDRSCIHLEFDISRTGITYETG DHVGVYAENHVEIVEEAGKLLGHSLDLVFSIHADKEDGSPLESAVPPPFPGPCTLGTGLA RYADLLNPPRKSALVALAAYATEPSEAEKLKHLTSPDGKDEYSQWIVASQRSLLEVMAAF PSAKPPLGVFFAAIAPRLQPRYYSISSSPRLAPSRVHVTSALVYGPTPTGRIHKGVCSTW MKNAVPAEKSHECSGAPIFIRASNFKLPSNPSTPIVMVGPGTGLAPFRGFLQERMALKED GEELGSSLLFFGCRNRQMDFIYEDELNNFVDQGVISELIMAFSREGAQKEYVQHKMMEKA AQVWDLIKEEGYLYVCGDAKGMARDVHRTLHTIVQEQEGVSSSEAEAIVKKLQTEGRYLR DVW A. thaliana AtCPR2 SEQ ID NO: 29MASSSSSSSTSMIDLMAAIIKGEPVIVSDP ANASAYESVAAELSSMLIENRQFAMIVTTSIAVLIGCIVMLVWRRSGSGNSKRVEPLKPL VIKPREEEIDDGRKKVTIFFGTQTGTAEGFAKALGEEAKARYEKTRFKIVDLDDYAADDD EYEEKLKKEDVAFFFLATYGDGEPTDNAARFYKWFTEGNDRGEWLKNLKYGVFGLGNRQY EHFNKVAKVVDDILVEQGAQRLVQVGLGDDDQCIEDDFTAWREALWPELDTILREEGDTA VATPYTAAVLEYRVSIHDSEDAKFNDINMANGNGYTVFDAQHPYKANVAVKRELHTPESD RSCIHLEFDIAGSGLTYETGDHVGVLCDNLSETVDEALRLLDMSPDTYFSLHAEKEDGTP ISSSLPPPFPPCNLRTALTRYACLLSSPKKSALVALAAHASDPTEAERLKHLASPAGKDE YSKWVVESQRSLLEVMAEFPSAKPPLGVFFAGVAPRLQPRFYSISSSPKIAETRIHVTCA LVYEKMPTGRIHKGVCSTWMKNAVPYEKSENCSSAPIFVRQSNFKLPSDSKVPIIMIGPG TGLAPFRGFLQERLALVESGVELGPSVLFFGCRNRRMDFIYEEELQRFVESGALAELSVA FSREGPTKEYVQHKMMDKASDIWNMISQGAYLYVCGDAKGMARDVHRSLHTIAQEQGSMD STKAEGFVKNLQTSGRYLRDVW A. thaliana eATR2SEQ ID NO: 30 MASSSSSSSTSMIDLMAAIIKGEPVIVSDPANASAYESVAAELSSMLIENRQFAMIVTTS IAVLIGCIVMLVWRRSGSGNSKRVEPLKPLVIKPREEEIDDGRKKVTIFFGTQTGTAEGF AKALGEEAKARYEKTRFKIVDLDDYAADDDEYEEKLKKEDVAFFFLATYGDGEPTDNAAR FYKWFTEGNDRGEWLKNLKYGVFGLGNRQYEHFNKVAKVVDDILVEQGAQRLVQVGLGDD DQCIEDDFTAWREALWPELDTILREEGDTAVATPYTAAVLEYRVSIHDSEDAKFNDITLA NGNGYTVFDAQHPYKANVAVKRELHTPESDRSCIHLEFDIAGSGLTMKLGDHVGVLCDNL SETVDEALRLLDMSPDTYFSLHAEKEDGTPISSSLPPPFPPCNLRTALTRYACLLSSPKK SALVALAAHASDPTEAERLKHLASPAGKDEYSKWVVESQRSLLEVMAEFPSAKPPLGVFF AGVAPRLQPRFYSISSSPKIAETRIHVTCALVYEKMPTGRIHKGVCSTWMKNAVPYEKSE KLFLGRPIFVRQSNFKLPSDSKVPIIMIGPGTGLAPFRGFLQERLALVESGVELGPSVLF FGCRNRRMDFIYEEELQRFVESGALAELSVAFSREGPTKEYVQHKMMDKASDIWNMISQG AYLYVCGDAKGMARDVHRSLHTIAQEQGSMDSTKAEGFVKNLQTSGRYLRDVW S. rebaudiana SrCPR3 SEQ ID NO: 32MAQSNSVKISPLDLVTALFSGKVLDTSNAS ESGESAMLPTIAMIMENRELLMILTTSVAVLIGCVVVLVWRRSSTKKSALEPPVIVVPKR VQEEEVDDGKKKVTVFFGTQTGTAEGFAKALVEEAKARYEKAVFKVIDLDDYAADDDEYE EKLKKESLAFFFLATYGDGEPTDNAARFYKWFTEGDAKGEWLNKLQYGVFGLGNRQYEHF NKIAKVVDDGLVEQGAKRLVPVGLGDDDQCIEDDFTAWKELVWPELDQLLRDEDDTTVAT PYTAAVAEYRVVFHEKPDALSEDYSYTNGHAVHDAQHPCRSNVAVKKELHSPESDRSCTH LEFDISNTGLSYETGDHVGVYCENLSEVVNDAERLVGLPPDTYFSIHTDSEDGSPLGGAS LPPPFPPCTLRKALTCYADVLSSPKKSALLALAAHATDPSEADRLKFLASPAGKDEYSQW IVASQRSLLEVMEAFPSAKPSLGVFFASVAPRLQPRYYSISSSPKMAPDRIHVTCALVYE KTPAGRIHKGVCSTWMKNAVPMTESQDCSWAPIYVRTSNFRLPSDPKVPVIMIGPGTGLA PFRGFLQERLALKEAGTDLGLSILFFGCRNRKVDFIYENELNNFVETGALSELIVAFSRE GPTKEYVQHKMSEKASDIWNLLSEGAYLYVCGDAKGMAKDVHRTLHTIVQEQGSLDSSKA ELYVKNLQMSGRYLRDVW Artemisia annua AaCPRSEQ ID NO: 33 MAQSTTSVKLSPFDLMTALLNGKVSFDTSNTSDTNIPLAVPMENRELLMILTTSVAVLIG CVVVLVWRRSSSAAKKAAESPVIVVPKKVTEDEVDDGRKKVIVFFGTQTGTAEGFAKALV EEAKARYEKAVFKVIDLDDYAAEDDEYEEKLKKESLAFFFLATYGDGEPTDNAARFYKWF TEGEEKGEWLDKLQYAVFGLGNRQYEHFNKIAKVVDEKLVEQGAKRLVPVGMGDDDQCIE DDFTAWKELVWPELDQLLRDEDDTSVATPYTAAVAEYRVVFHDKPETYDQDQLTNGHAVH DAQHPCRSNVAVKKELHSPLSDRSCTHLEFDISNTGLSYETGDHVGVYVENLSEVVDEAE KLIGLPPHTYFSVHADNEDGTPLGGASLPPPFPPCTLRKALASYADVLSSPKKSALLALA AHATDSTEADRLKFLASPAGKDEYAQWIVASHRSLLEVMEAFPSAKPPLGVFFASVAPRL QPRYYSISSSPRFAPNRIHVTCALVYEQTPSGRVHKGVCSTWMKNAVPMTESQDCSWAPI YVRTSNFRLPSDPKVPVIMIGPGTGLAPFRGFLQERLAQKEAGTELGTAILFFGCRNRKV DFIYEDELNNFVETGALSELVTAFSREGATKEYVQHKMTQKASDIWNLLSEGAYLYVCGD AKGMAKDVHRTLHTIVQEQGSLDSSKAELYVKNLQMAGRYLRDVW Pelargonium graveolens PgCPR SEQ ID NO: 34MAQSSSGSMSPFDPMTAIIKGKMEPSNASL GAAGEVTAMILDNRELVMILTTSIAVLIGCVVVFIWRRSSSQTPTAVQPLKPLLAKETES EVDDGKQKVTIFFGTQTGTAEGFAKALADEAKARYDKVTFKVVDLDDYAADDEEYEEKLK KETLAFFFLATYGDGEPTDNAARFYKWFLEGKERGEWLQNLKFGVFGLGNRQYEHFNKIA IVVDEILAEQGGKRLISVGLGDDDQCIEDDFTAWRESLWPELDQLLRDEDDTTVSTPYTA AVLEYRVVFHDPADAPTLEKSYSNANGHSVVDAQHPLRANVAVRRELHTPASDRSCTHLE FDISGTGIAYETGDHVGVYCENLAETVEEALELLGLSPDTYFSVHADKEDGTPLSGSSLP PPFPPCTLRTALTLHADLLSSPKKSALLALAAHASDPTEADRLRHLASPAGKDEYAQWIV ASQRSLLEVMAEFPSAKPPLGVFFASVAPRLQPRYYSISSSPRIAPSRIHVTCALVYEKT PTGRVHKGVCSTWMKNSVPSEKSDECSWAPIFVRQSNFKLPADAKVPIIMIGPGTGLAPF RGFLQERLALKEAGTELGPSILFFGCRNSKMDYIYEDELDNFVQNGALSELVLAFSREGP TKEYVQHKMMEKASDIWNLISQGAYLYVCGDAKGMARDVHRTLHTIAQEQGSLDSSKAES MVKNLQMSGRYLRDVWCamptotheca acuminata cytochrome P450 reductase; CaCPR SEQ ID NO: 53MAQSSSVKVSTFDLMSAILRGRSMDQTNVS FESGESPALAMLIENRELVMILTTSVAVLIGCFVVLLWRRSSGKSGKVTEPPKPLMVKTE PEPEVDDGKKKVSIFYGTQTGTAEGFAKALAEEAKVRYEKASFKVIDLDDYAADDEEYEE KLKKETLTFFFLATYGDGEPTDNAARFYKWFMEGKERGDWLKNLHYGVFGLGNRQYEHFN RIAKVVDDTIAEQGGKRLIPVGLGDDDQCIEDDFAAWRELLWPELDQLLQDEDGTTVATP YTAAVLEYRVVFHDSPDASLLDKSFSKSNGHAVHDAQHPCRANVAVRRELHTPASDRSCT HLEFDISGTGLVYETGDHVGVYCENLIEVVEEAEMLLGLSPDTFFSIHTDKEDGTPLSGS SLPPPFPPCTLRRALTQYADLLSSPKKSSLLALAAHCSDPSEADRLRHLASPSGKDEYAQ WVVASQRSLLEVMAEFPSAKPPIGAFFAGVAPRLQPRYYSISSSPRMAPSRIHVTCALVF EKTPVGRIHKGVCSTWMKNAVPLDESRDCSWAPIFVRQSNFKLPADTKVPVLMIGPGTGL APFRGFLQERLALKEAGAELGPAILFFGCRNRQMDYIYEDELNNFVETGALSELIVAFSR EGPKKEYVQHKMMEKASDIWNMISQEGYIYVCGDAKGMARDVHRTLHTIVQEQGSLDSSK TESMVKNLQMNGRYLRDVW Alcohol DehydrogenaseBrachypodium distachyon BdDH SEQ ID NO: 35MSAAAAVSSSSSPRLEGKVALVTGGASGIG EAIVRLFRQHGAKVCIADVQDEAGQQVRDSLGDDAGTDVLFVHCDVTVEEDVSRAVDAAA EKFGTLDIMVNNAGITGDKVTDIRNLDFAEVRKVFDINVHGMLLGMKHAARVMIPGKKGS IVSLASVASVMGGMGPHAYTASKHAVVGLTKSVALELGKHGIRVNCVSPYAVPTALSMPH LPQGEHKGDAVRDFLAFVGGEANLKGVDLLPKDVAQAVLYLASDEARYISALNLVVDGGF TSVNPNLKAFED Citrus sinensis CsABA2SEQ ID NO: 36 MSNSNSTDSSPAVQRLVGRVALITGGATGIGESTVRLFHKHGAKVCIADVQDNLGQQVCQ SLGGEPDTFFCHCDVTKEEDVCSAVDLTVEKFGTLDIMVNNAGISGAPCPDIREADLSEF EKVFDINVKGVFHGMKHAARIMIPQTKGTIISICSVAGAIGGLGPHAYTGSKHAVLGLNK NVAAELGKYGIRVNCVSPYAVATGLALAHLPEEERTEDAMVGFRNFVARNANMQGTELTA NDVANAVLFLASDEARYISGTNLMVDGGFT SVNHSLRVFRCitrus sinensis CsDH SEQ ID NO: 37 MATPPISSLISQRLLGKVALVTGGASGIGEGIVRLFHRHGAKVCFVDVQDELGYRLQESL VGDKDSNIFYSHCDVTVEDDVRRAVDLTVTKFGTLDIMVNNAGISGTPSSDIRNVDVSEF EKVFDINVKGVFMGMKYAASVMIPRKQGSIISLGSVGSVIGGIGPHHYISSKHAVVGLTR SIAAELGQHGIRVNCVSPYAVPTNLAVAHLPEDERTEDMFTGFREFAKKNANLQGVELTV EDVANAVLFLASEDARYISGDNLIVDGGFT RVNHSFRVFRCitrus sinensis CsDH1 SEQ ID NO: 38 MSKPRLQGKVAIIMGAASGIGEATAKLFAEHGAFVIIADIQDELGNQVVSSIGPEKASYR HCDVRDEKQVEETVAYAIEKYGSLDIMYSNAGVAGPVGTILDLDMAQFDRTIATNLAGSV MAVKYAARVMVANKIRGSIICTTSTASTVGGSGPHAYTISKHGLLGLVRSAASELGKHGI RVNCVSPFGVATPFSAGTINDVEGFVCKVANLKGIVLKAKHVAEAALFLASDESAYVSGH DLVVDGGFTAVTNVMSMLEGHGCitrus sinensis CsDH2 SEQ ID NO: 39 MSNPRMEGKVALITGAASGIGEAAVRLFAEHGAFVVAADVQDELGHQVAASVGTDQVCYH HCDVRDEKQVEETVRYTLEKYGKLDVLFSNAGIMGPLTGILELDLTGFGNTMATNVCGVA ATIKHAARAMVDKNIRGSIICTTSVASSLGGTAPHAYTTSKHALVGLVRTACSELGAYGI RVNCISPFGVATPLSCTAYNLRPDEVEANSCALANLKGIVLKAKHIAEAALFLASDESAY ISGHNLAVDGGFTVVNHSSSSATCitrus sinensis CsDH3 SEQ ID NO: 40 MTTAGSRDSPLVAQRLLGKVALVTGGATGIGESIVRLFHKHGAKVCVVDINDDLGQHLCQ TLGPTTRFIHGDVAIEDDVSRAVDFTVANFGTLDIMVNNAGMGGPPCPDIREFPISTFEK VFDINTKGTFIGMKHAARVMIPSKKGSIVSISSVISAIGGAGPHAYTASKHAVLGLIKSV AAELGQHGIRVNCVSPYAILTNLALAHLHEDERTDDARAGFRAFIGKNANLQGVDLVEDD VANAVLFLASDDARYISGDNLFVDGGFTCT NHSLRVFRRhodococcus erythropolis ReCDH SEQ ID NO: 41MARVEGQVALITGAARGQGRSHAIKLAEEG ADVILVDVPNDVVDIGYPLGTADELDQTAKDVENLGRKAIVIHADVRDLESLTAEVDRAV STLGRLDIVSANAGIASVPFLSHDIPDNTWRQMIDINLTGVWHTAKVAVPHILAGERGGS IVLTSSAAGLKGYAQISHYSAAKHGVVGLMRSLALELAPHRVRVNSLHPTQVNTPMIQNE GTYRIFSPDLENPTREDFEIASTTTNALPIPWVESVDVSNALLFLVSEDARYITGAAIPV DAGTTLK VoDH1 SEQ ID NO: 42MSTASSGDVSLLSQRLVGKVALITGGATGI GESIARLFYRHGAKVCIVDIQDNPGQNLCRELGTDDACFFHCDVSIEIDVIRAVDFVVNR FGKLDIMVNNAGIADPPCPDIRNTDLSIFEKVFDVNVKGTFQCMKHAARVMVPQKKGSII SLTSVASVIGGAGPHAYTGSKHAVLGLTKSVAAELGLHGIRVNCVSPYAVPTGMPLAHLP ESEKTEDAMMGMRAFVGRNANLQGIELTVDDVANSVVFLASDEARYVSGLNLMLDGGFSC VNHSLRVFR Vitis vinifera VvDHSEQ ID NO: 43 MAATSIDNSPLPSQRLLGKVALVTGGATGIGESIVRLFLKQGAKVCIVDVQDDLGQKLCD TLGGDPNVSFFHCDVTIEDDVCHAVDFTVTKFGTLDIMVNNAGMAGPPCSDIRNVEVSMF EKVFDVNVKGVFLGMKHAARIMIPLKKGTIISLCSVSSAIAGVGPHAYTGSKCAVAGLTQ SVAAEMGGHGIRVNCISPYAIATGLALAHLPEDERTEDAMAGFRAFVGKNANLQGVELTV DDVAHAAVFLASDEARYISGLNLMLDGGFS CTNHSLRVFRZingiber zerumbet ZzSDR SEQ ID NO: 44 MRLEGKVALVTGGASGIGESIARLFIEHGAKICIVDVQDELGQQVSQRLGGDPHACYFHC DVTVEDDVRRAVDFTAEKYGTIDIMVNNAGITGDKVIDIRDADFNEFKKVFDINVNGVFL GMKHAARIMIPKMKGSIVSLASVSSVIAGAGPHGYTGAKHAVVGLTKSVAAELGRHGIRV NCVSPYAVPTRLSMPYLPESEMQEDALRGFLTFVRSNANLKGVDLMPNDVAEAVLYLATE ESKYVSGLNLVIDGGFSIANHTLQVFE

What is claimed is:
 1. A microbial host cell for producing rotundone,the microbial cell expressing a heterologous α-guaiene synthase enzyme(αGTPS) and a heterologous α-guaiene oxidase (αGOX) enzyme.
 2. Themicrobial cell of claim 1, further expressing a farnesyl diphosphatesynthase.
 3. The microbial cell of claim 2, wherein the αGTPS enzymecomprises an amino acid sequence of any one of SEQ ID NOs: 1 to 21, orvariant thereof.
 4. The microbial cell of claim 3, wherein α-guaienesynthase enzyme comprises an amino acid sequence having 50% or moresequence identity with any one of SEQ ID NOs: 1 to
 21. 5. The microbialcell of claim 3, wherein the αGTPS enzyme comprises an amino acidsequence having 50% or more sequence identity to SEQ ID NO:
 8. 6. Themicrobial cell of claim 5, wherein the αGTPS enzyme comprises one ormore amino acid substitutions at positions selected from 72, 273, 290,368, 371, 374, 377, 381, 382, 399, 406, 419, 433, 442, 443, 454, 512,and 522 relative to SEQ ID NO:
 8. 7. The microbial cell of claim 6,wherein the αGTPS enzyme comprises one or more amino acid substitutionsselected from T72I, M273L, R290K, F368M, I371L, S374A, R377V, Y381W,F382L, I399V, F406L, L419T, V433I, Y442L, I443M, E454K, F512L, and K522Drelative to SEQ ID NO:
 8. 8. The microbial cell of claim 4, wherein theα-guaiene synthase produces predominantly α-guaiene as the product fromFPP substrate.
 9. The microbial cell of any one of claims 1 to 8,wherein the αGOX enzyme is a cytochrome P450 (CYP450) enzyme.
 10. Themicrobial cell of claim 9, wherein the CYP450 comprises the amino acidsequence of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 22, SEQ ID NO: 23,SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or a variant thereof. 11.The microbial cell of claim 10, wherein the CYP450 comprises an aminoacid sequence that has 50% or more sequence identity with any one of SEQID NOs: 51, 52, or 22 to
 26. 12. The microbial cell of claim 9, whereinthe CYP450 comprises an amino acid sequence having 50% or more sequenceidentity to SEQ ID NO:
 51. 13. The microbial cell of claim 9, whereinthe CYP450 comprises an amino acid sequence having 50% or more sequenceidentity to SEQ ID NO:
 52. 14. The microbial cell of any one of claims 1to 13, wherein the microbial host cell expresses a cytochrome P450reductase enzyme.
 15. The microbial cell of any one of claims 1 to 14,wherein the αGTPS and αGOX are expressed together in an operon.
 16. Themicrobial cell of any of claims 1 to 15, wherein the microbial host cellfurther expresses one or more alcohol dehydrogenases (ADHs).
 17. Themicrobial cell of claim 16, wherein the ADH comprises an amino acidsequence of any one of SEQ ID NOs: 35-44, or a variant thereof.
 18. Themicrobial cell of claim 11, wherein the ADH comprises an amino acidsequence having 50% or more sequence identity to SEQ ID NO:
 43. 19. Themicrobial cell of any one of claims 1 to 18, wherein one or more enzymesare expressed from extrachromosomal elements.
 20. The microbial cell ofany one of claims 1 to 18, wherein one or more enzymes are expressedfrom genes that are chromosomally integrated.
 21. The microbial cell ofany one of claims 1 to 20, wherein the microbial host cell overexpressesone or more enzymes in the methylerythritol phosphate (MEP) or themevalonic acid (MVA) pathway.
 22. The microbial cell of any one ofclaims 1 to 21, wherein the microbial cell is a bacteria, optionallyselected from Escherichia spp., Bacillus spp., Corynebacterium spp.,Rhodobacter spp Zymomonas spp., Vibrio spp., and Pseudomonas spp. 23.The microbial cell of claim 22, wherein the bacterial host cell isselected from Escherichia coli, Bacillus subtilis, Corynebacteriumglutamicum, Rhodobacter capsulatus, Rhodobacter sphaeroides, Zymomonasmobilis, Vibrio natriegens, or Pseudomonas putida.
 24. The microbialcell of any one of claims 1 to 21, wherein the microbial host cell is ayeast, optionally selected from Saccharomyces, Pichia, or Yarrowia. 25.The microbial cell of claim 24, wherein the microbial cell isSaccharomyces cerevisiae, Pichia pastoris, and Yarrowia lipolytica. 26.A method for making rotundone, comprising: culturing the microbial cellof any one of claims 1 to 25, and recovering the rotundone.
 27. Themethod of claim 26, wherein the microbial cells are cultured with C1,C2, C3, C4, C5, and/or C6 carbon substrates.
 28. The method of claim 27,wherein the carbon source is glucose, sucrose, fructose, xylose, and/orglycerol.
 29. The method of any one of claims 26 to 28, wherein cultureconditions are selected from aerobic, microaerobic, and anaerobic. 30.The method of claim 29, wherein the microbial cell is cultured at atemperature between 22° C. and 37° C.
 31. A method for producingrotundone, comprising feeding α-guaiene to a microbial cell expressingan α-guaiene oxidase (αGOX), or to an extract of the cell, or to areaction vessel comprising recombinant αGOX, wherein the αGOX optionallycomprises the amino acid sequence of SEQ ID NO: 51, SEQ ID NO: 52, SEQID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26,or a variant thereof.
 32. The method of claim 31, wherein the αGOX is aCYP450 comprising an amino acid sequence that has 50% or more sequenceidentity with any one of SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NOS: 22to
 26. 33. The method of claim 31, wherein the αGOX is a non-heme ironoxygenase (NHIO) or a laccase.
 34. The method of any one of claims 31 to33, wherein the microbial cell expresses one or more alcoholdehydrogenases.
 35. The method of any one of claims 31 to 33, whereinthe extract or reaction vessel further comprises one or more alcoholdehydrogenases.
 36. The method of claim 33 or 34, wherein the alcoholdehydrogenase comprises an amino acid sequence selected from SEQ ID NOs:35-44, or a variant thereof.
 37. The method of any one of claims 31 to36, further comprising, recovering rotundone.